Anti-Axl antagonistic antibodies

ABSTRACT

Described are antibodies that specifically bind to the Axl protein and inhibit the interaction between Axl and the Axl-ligand, Gas6. Also disclosed are methods for the production and use of the anti-Axl antibodies.

This application is a continuation of U.S. application Ser. No.15/533,049, filed Jun. 5, 2017, which is a national phase entry pursuantto 35 U.S.C. § 371 of International Application No. PCT/EP2015/080654,filed Dec. 18, 2015, which claims the benefit of priority of GreatBritain Application No. 1422605.4, filed Dec. 18, 2014, each of which isincorporated by reference herein in its entirety for any purpose.

The present disclosure relates to antibodies that specifically bind tothe Axl protein. Also disclosed are methods for the production and useof the anti-Axl antibodies.

BACKGROUND

Axl is a member of the TAM (Tyro3-Axl-Mer) receptor tyrosine kinases(RTK) that share the vitamin K-dependent ligand Gas6 (growtharrest-specific 6). TAM family RTKs regulate a diverse range of cellularresponses including cell survival, proliferation, autophagy, migration,angiogenesis, platelet aggregation, and natural killer celldifferentiation. Axl is expressed in many embryonic tissues and isthought to be involved in mesenchymal and neural development, withexpression in adult tissues largely restricted to smooth muscle cells(MGI Gene Expression Database; www.informatics.jax.org). Axl activationis linked to several signal transduction pathways, including Aid, MAPkinases, NF-κB, STAT, and others.

Originally identified as a transforming gene from a patient with chronicmyelogenous leukaemia, Axl has since been associated with varioushigh-grade cancers and correlated with poor prognosis.

Axl receptor overexpression has been detected in a wide range of solidtumours and myeloid leukaemia (Linger et al, Adv Cancer Res. 100: 35,2008; Linger et al, Expert Opin Ther Targets. 14:1073, 2010).

Axl expression correlates with malignant progression and is anindependent predictor of poor patient overall survival in severalmalignancies including pancreatic (Song et al, Cancer. 117:734, 2011),prostate (Paccez et al, Oncogene. 32:698, 2013), lung (Ishikawa et al.Ann Surg Oncol. 2012; Zhang et al, Nat Genet. 44:852, 2012), breast(Gjerdrum, Proc natl Acad Sci USA 107:1124, 2010), colon cancer (Yuen etal, PLoS One, 8:e54211, 2013) and acute myeloid leukaemia (AML)(Ben-Batalla et al, Blood 122:2443, 2013).

Axl signal transduction is activated by a protein ligand (Gas6) secretedby tumour associated macrophages (Loges et al, Blood. 115:2264, 2010) orautocrine mechanisms (Gjerdrum, Proc natl Acad Sci USA 107:1124, 2010),that drives receptor dimerization, autophosphorylation and downstreamsignalling, such as via PI3 kinase (PI3K)-AKT, particularly AKT andmitogen-activated protein kinase (MAPK) pathways (Korshunov, ClinicalScience. 122:361, 2012). Heterodimerization with other tyrosine kinasereceptors, e.g. epidermal growth factor receptor (EGFR), is alsoreported to occur (Linger et al, Expert Opin Ther Targets. 14:1073,2010; Meyer et al Science Signalling 6:ra66, 2013).

Aberrant activation of Axl in tumour cells is widely associated withacquired drug resistance to targeted therapeutics in vitro and in vivo(Zhang et al. Nat Genet. 44: 852, 2012; Byers et al. Clin Cancer Res.19: 279, 2013). Axl-targeting agents block tumour formation, metastasisand reverse drug resistance (e.g. to erlotinib) by reversing EMT/CSCcharacteristics in several experimental cancer models, including triplenegative breast cancer, hormone resistant prostate cancer andadenocarcinoma of the lung (Holland et al Cancer Res 70:1544, 2010;Gjerdrum, Proc natl Acad Sci USA 107:1124, 2010; Zhang et al. Nat Genet.44: 852, 2012; Paccez et al, Oncogene. 32:698, 2013).

Other applications relating to Axl and anti-Axl antibodies includeEP2267454A2 [Diagnosis and prevention of cancer cell invasion measuring. . . Axl—Max Planck]; WO2009063965 [anti Axl—Chugai Pharmaceutical];WO2011159980A1 [anti-Axl—Genentech], WO2011014457A1 [combinationtreatments Axl and VEGF antagonists—Genentech]; WO2012-175691A1 [AntiAxl 20G7-D9—INSERM], WO2012-175692A1 [Anti Axl 3E3E8—INSERM];WO2009/062690A1 [anti Axl—U3 Pharma] and WO2010/130751A1 [humanised antiAxl—U3 Pharma].

In view of the role of Axl in tumourigenesis, it is desirable toidentify further antibodies with advantageous properties thatspecifically bind Axl. The present disclosure concerns such antibodies.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1

Overlay plots of sensograms from the binding analyses showinginteractions of MAbs 10C9 and 10G5 with recombinant human (rh) Axl,rhMer and rhTyro3. The curves after subtraction of blank surface signalsare shown.

FIG. 2

Biacore analyses of ligands (MAb 10C9, MAb 10G5 and rmGas6) interactingwith a sensor chip CM5 coated with rhAxl, recombinant murine (rm) Axland rhTyro3. The curves after subtraction of blank surface signals areshown.

FIG. 3

Biacore analyses of ligands (MAbs 10C9 and 10G5) interacting with asensor chip CM5 coated with recombinant human Axl (rhAxl) and Axlantigen from cynomolgus monkey (cyno-Axl). The curves after subtractionof blank surface signals are shown.

FIG. 4

Kinetic analysis of MAbs 10C9 and 10G5 interacting with rhAxlimmobilized on the surface of the Biacore sensor chip. Overlay plots ofsensograms for different antibody concentrations (1.3-666.7 nM for 10C9and 0.3-166.7 nM for 10G5) are shown. The precise kinetic analysis wasperformed using BIA evaluation software and curve fitting according to1:1 Langmuir binding model. The affinity constants (kinetic and steadystate) as well as the calculated half-live of antigen binding at 25° C.are shown in Table 1, below.

TABLE 1 On-rate Off-rate Half-life MAb (k_(on); M⁻¹s⁻¹) (k_(off); s⁻¹)K_(D) (M) (t_(1/2); min) 10C9 1.61 × 10⁶ 2.89 × 10⁻⁴ 1.80 × 10⁻¹⁰ 39.97min 10G5 8.29 × 10⁵ 4.39 × 10⁻⁴ 5.30 × 10⁻¹⁰ 26.32 min

FIG. 5

Analysis of the competition between MAbs 10C9 or 10G5 (1st sample) andanti-Axl MAb MAB154 (R&D Systems), antibodies 10C9 and 10G5, rhGas6 andrmGas6 (2nd samples) using Biacore 3000. The overlay plot of sensogramsusing different 2nd samples is shown. Start points of injections of the1st sample (10C9 or 10G5) and the 2nd sample are indicated with arrows.

FIG. 6

The effect of anti-Axl antibodies on the development onthree-dimensional (3D) organotypic tumour masses. Highly aggressivehuman mammary carcinoma cells MDA-MB-231 were treated with eithercontrol IgG (shown in the middle upper panel) or anti-Axl MAbs (lowerpanels) while growing in the presence of extracellular matrix, thuscreating 3D organotypic models. As positive control, MDA-MB-231 cellswith knocked-down Axl expression are shown.

FIG. 7

Effect of anti-Axl antibodies 10C9 and 10G5 on established 3Dorganotypic tumour masses. The developed 9-days old stellate-shaped 3Dorganoid masses of human mammary carcinoma cells (MDA-MB-231) weretreated with either control IgG or anti-Axl antibodies 10C9 and 10G5 for72 hours. Images were captured using bright field; arrows indicateapoptotic, degrading stellate-shaped cells.

FIG. 8

Western blot analysis illustrating effect of treatment with eitherantibodies of multikinase inhibitor Foretinib on Axl receptorexpression. Highly aggressive human mammary carcinoma cells MDA-MB-231were treated with either antibodies (irrelevant IgG control and anti-AxlMAbs 10C9, 10G5 and MAb #3) or Foretinib for 24 hrs before loading onSDS-PAA gel. The levels of actin protein were used as loading controls.

FIG. 9

Western blot analysis illustrating inhibition of Gas6-mediated Axlsignalling in the presence of mouse monoclonal antibodies 10C9 and 10G5.Phosphorylation of Aid on Ser⁴⁷³ was used as surrogate readout for Axlactivity. M, molecular weight markers. Immunoblots of total cell lysateswere probed with anti-phospho-Akt (Ser⁴⁷³), or anti-GAPDH(glyceraldehyde 3-phosphate dehydrogenase) as loading control.

FIG. 10

Amino acid sequences of the VH and VL domains derived from anti-Axlmonoclonal antibodies 10C9 (SEQ ID NOs: 3 and 4, respectively) and 10G5(SEQ ID NOs: 21 and 22, respectively). The CDR regions of the heavy andlight chains are underlined. The potential N-glycosylation site inCDR-HI of 10C9 VH domain is shown in bold.

Also included is the sequence of a 10G5 VH variant wherein the glutamine(Q) at position 1 of the VH domain is substituted with a glutamate (E);this variant is termed “10G5 [Q1E]” (SEQ ID NOs: 45 and 22).

FIG. 11

Dose-dependent binding of anti-Axl mouse antibody 10C9, its chimeric(mouse variable/human constant) counterpart (c10C9) and the chimericvariant of antibody 10G5 (c10G5) to Axl-positive cells. Differentconcentrations of mouse and chimeric antibodies were tested in flowcytometry for binding to triple-negative breast cancer cell lineMDA-MB-231. The bound mouse and chimeric antibodies were detected withAPC-conjugated donkey F(ab′)₂ fragments specific for either mouse IgG(H+L), 1:500 dilution, or human IgG (H+L), 1:300 dilution, respectively(both from Jackson ImmunoResearch). The cell staining was measured usingAccuri C6 flow cytometer (BD Biosciences). MFI, geometric meanfluorescence intensity.

FIG. 12

Overlay plots of sensograms from the Biacore binding analyses showinginteractions of the chimeric antibodies c10C9 and c10G5 and their murinecounterparts with recombinant human (rh) Axl. The curves aftersubtraction of blank surface signals are shown.

FIG. 13

Kinetic analysis of chimeric antibodies c10C9 and c10G5 interacting withrhAxl immobilized on the surface of the Biacore sensor chip. Overlayplots of sensograms for different antibody concentrations (1.3-666.7 nMfor c10C9 and 0.3-166.7 nM for c10G5) are shown. The precise kineticanalysis was performed using BIA evaluation software and curve fittingaccording to 1:1 Langmuir binding model. The affinity constants (kineticand steady state) as well as the calculated half-live of antigen bindingat 25° C. are shown in Table 2, below.

TABLE 2 On-rate Off-rate Half-life MAb (k_(on); M⁻¹s⁻¹) (k_(off); s⁻¹)K_(D) (M) (t_(1/2); min) c10C9 2.16 × 10⁶ 2.19 × 10⁻⁴ 1.02 × 10⁻¹⁰ 52.75min c10G5 1.64 × 10⁶ 1.69 × 10⁻⁴ 1.03 × 10⁻¹⁰ 68.36 min

FIG. 14

Inhibition of A549 xenograft tumour growth by chimeric antibody 10G5.The antibody was administrated intraperitoneally at 20 mg/kg, twice aweek, starting when the mean tumour size reached 100 mm³. Tumour growthcurves for the groups treated with either vehicle (sterile PBS) orchimeric 10G5 are shown. Error bars represent standard error of the mean(SEM). Statistical analysis was performed using two-way ANOVA. **,P<0.01.

FIG. 15

Inhibition of Mv4-11 xenograft tumour growth by the chimeric antibody10G5. The antibody was administrated intraperitoneally at 30 mg/kg,twice a week, starting when the mean tumour size reached 200 mm³. Tumourgrowth curves for the groups treated with either vehicle (sterile PBS)or chimeric 10G5 are shown. Error bars represent standard error of themean (SEM). Statistical analysis was performed using two-way ANOVA. *,P<0.05; **, P<0.01; ****, P<0.0001.

FIG. 16

Data from Example 16. The antibody Glymax-c10G5 significantly attenuatedgrowth of A549 tumours compared with the c10G5 (P<0.0001, as determinedby two-way ANOVA). The significant difference in activity of wt anddefucosylated versions of the chimeric 10G5 indicates importance ofantibody-dependent cellular cytotoxicity (ADCC) in inhibition of tumorgrowth.

FIG. 17

Data from Example 17. The FV1 antibody significantly attenuated growthof A549 tumours compared with the control (P<0.051, as determined bytwo-way ANOVA); around 25% inhibition was observed after two weeks oftreatment.

FIG. 18

Data from Example 18. The FV2 antibody showed moderate anti-tumoractivity, similar to the anti-tumor effect of the Anti-EGFR therapeuticantibody cetuximab (Erbitux). Combination of both antibodies resulted insignificant tumor growth retardation (P<0.0001; as determined by two-wayANOVA) when compared to isotype control treated animals. The combinedeffect was also significant when compared to the groups treated witheither FV2 or Erbitux alone (P<0.05; as determined by two-way ANOVA).

DISCLOSURE OF THE INVENTION

This invention provides antibodies that bind to Axl protein and inhibitsthe binding of Axl to its ligand Gas6. The antibodies preferably alsodownregulate Axl expression, inhibit Axl receptor signalling, and/orinhibit tumour growth.

Disclosed herein are two specific examples of such antibodies that bindAxl and inhibit the binding of Axl to its ligand Gas6. These antibodiesare referred to herein as “10C9” (obtainable from hybridoma UT-10C9-B9,as described herein) and “10G5” (obtainable from hybridoma WR-10G5-E5,as described herein).

Accordingly, the present invention provides an antibody that binds tothe epitope bound by the 10C9 antibody obtainable from the hybridomaUT-10C9-B9, as described herein. Also provided is an antibody that bindsto the epitope bound by the 10G5 antibody obtainable from the hybridomaWR-10G5-E5, as described herein.

Preferably the antibody inhibits the binding of Axl to its ligand Gas6.Even more preferably, the antibody also downregulates Axl expression,inhibits Axl receptor signalling, and/or inhibits tumour growth.

Sequences

The following sequences are disclosed herein (see ‘SEQUENCES’ sectionbelow for full sequence):

SEQ ID NO.1→10C9 VH encoding nucleotide sequence

SEQ ID NO.2→10C9 VL encoding nucleotide sequence

SEQ ID NO.3→10C9 VH encoding amino acid sequence

SEQ ID NO.4→10C9 VL encoding amino acid sequence

SEQ ID NO.5→10C9 VH CDR1 encoding amino acid sequence

SEQ ID NO.6→10C9 VH CDR2 encoding amino acid sequence

SEQ ID NO.7→10C9 VH CDR3 encoding amino acid sequence

SEQ ID NO.8→10C9 VL CDR1 encoding amino acid sequence

SEQ ID NO.9→10C9 VL CDR2 encoding amino acid sequence

SEQ ID NO.10→10C9 VL CDR3 encoding amino acid sequence

SEQ ID NO.11→10C9 VH FR1 encoding amino acid sequence

SEQ ID NO.12→10C9 VH FR2 encoding amino acid sequence

SEQ ID NO.13→10C9 VH FR3 encoding amino acid sequence

SEQ ID NO.14→10C9 VH FR4 encoding amino acid sequence

SEQ ID NO.15→10C9 VL FR1 encoding amino acid sequence

SEQ ID NO.16→10C9 VL FR2 encoding amino acid sequence

SEQ ID NO.17→10C9 VL FR3 encoding amino acid sequence

SEQ ID NO.18→10C9 VL FR4 encoding amino acid sequence

SEQ ID NO.19→10G5 VH encoding nucleotide sequence

SEQ ID NO.20→10G5 VL encoding nucleotide sequence

SEQ ID NO.21→10G5 VH encoding amino acid sequence

SEQ ID NO.22→10G5 VL encoding amino acid sequence

SEQ ID NO.23→10G5 VH CDR1 encoding amino acid sequence

SEQ ID NO.24→10G5 VH CDR2 encoding amino acid sequence

SEQ ID NO.25→10G5 VH CDR3 encoding amino acid sequence

SEQ ID NO.26→10G5 VL CDR1 encoding amino acid sequence

SEQ ID NO.27→10G5 VL CDR2 encoding amino acid sequence

SEQ ID NO.28→10G5 VL CDR3 encoding amino acid sequence

SEQ ID NO.29→10G5 VH FR1 encoding amino acid sequence

SEQ ID NO.30→10G5 VH FR2 encoding amino acid sequence

SEQ ID NO.31→10G5 VH FR3 encoding amino acid sequence

SEQ ID NO.32→10G5 VH FR4 encoding amino acid sequence

SEQ ID NO.33→10G5 VL FR1 encoding amino acid sequence

SEQ ID NO.34→10G5 VL FR2 encoding amino acid sequence

SEQ ID NO.35→10G5 VL FR3 encoding amino acid sequence

SEQ ID NO.36→10G5VL FR4 encoding amino acid sequence

SEQ ID NO.37→Human Axl encoding amino acid sequence

SEQ ID NO.38→Murine Ax encoding amino acid sequence

SEQ ID NO.39→Human Tyro3 encoding amino acid sequence

SEQ ID NO.40→Human Mer encoding amino acid sequence

SEQ ID NO.41→Human Akt3 encoding amino acid sequence

SEQ ID NO.42→Human Gas6 encoding amino acid sequence

SEQ ID NO.43→‘Cyno-Axl’ encoding amino acid sequence

SEQ ID NO.44→Linker

SEQ ID NO.45→10G5[QIE] VH encoding amino acid sequence

The 10C9 Antibody

In one aspect, the present invention provides an isolated antibody thatbinds Axl and that comprises the 10C9 VH domain (SEQ ID NO: 3) and/orthe 10C9 VL domain (SEQ ID NO: 4). Preferably the bound Axl is humanAxl.

In one aspect, the present invention provides an isolated antibody thatbinds Axl and that comprises the 10G5 VH domain (SEQ ID NO: 21) and/orthe 10G5 VL domain (SEQ ID NO: 22). Preferably the bound Axl is humanAxl. In an alternative non-preferred aspect, the present inventionprovides an isolated antibody that binds Axl and that comprises the10G5[Q1E] VH domain (SEQ ID NO: 45) and/or the 10G5 VL domain (SEQ IDNO: 22). Preferably the bound Axl is human Axl.

Generally, a VH domain is paired with a VL domain to provide an antibodyantigen binding site, although as discussed further below a VH domainalone may be used to bind antigen. In one preferred embodiment, the 10C9VH domain (SEQ ID NO: 3) is paired with the 10C9 VL domain (SEQ ID NO:4), so that an antibody antigen binding site is formed comprising boththe 10C9 VH and VL domains.

In other embodiments, the 10C9 VH is paired with a VL domain other thanthe 10C9 VL; light-chain promiscuity is well established in the art. Forexample, in some embodiments the 10C9 VH domain (SEQ ID NO: 3) is pairedwith the 10G5 VL domain (SEQ ID NO: 22).

One or more CDR's may be taken from the 10C9 VH or VL domain andincorporated into a suitable framework. This is discussed further below.10C9 VH CDR's 1, 2 and 3 are shown in SEQ ID Nos 5, 6 and 7,respectively. 10C9 VL CDR's 1, 2 and 3 are shown in SEQ ID Nos 8, 9, and10, respectively.

In one aspect of the invention there is provided an antibody that bindsAxl and that comprises:

-   -   an antibody VH domain selected from the group consisting of the        10C9 VH domain (SEQ ID NO.3) and a VH domain comprising a VH        CDR3 with the amino acid sequence of SEQ ID NO.7 and optionally        one or more VH CDR's with an amino acid sequence selected from        SEQ ID NO.6 and SEQ ID NO.5; and/or    -   an antibody VL domain selected from the group consisting of the        10C9 VL domain (SEQ ID NO. 4) and a VL domain comprising one or        more VL CDR's with an amino acid sequence selected from SEQ ID        NO.8, SEQ ID NO.9 and SEQ ID NO.10.

For example, the antibody may comprise an antibody VH domain comprisingthe VH CDR's with the amino acid sequences of SEQ ID NO.5, SEQ ID NO.6and SEQ ID NO.7. The antibody may further comprise an antibody VL domaincomprising the VL CDR's with the amino acid sequences of SEQ ID NO.8,SEQ ID NO.9 and SEQ ID NO.10.

In some embodiments the antibody comprises: (i) an antibody VH domaincomprising the VH CDR's with the amino acid sequences of SEQ ID NO.5,SEQ ID NO.6 and SEQ ID NO.7, and (ii) an antibody VL domain comprisingthe VL CDR's with the amino acid sequences of SEQ ID NO.8, SEQ ID NO.9and SEQ ID NO.10.

The antibody may comprise the 10C9 VH domain (SEQ ID NO. 3) and,optionally, further comprise the 10C9 VL domain (SEQ ID NO. 4)

Preferably the antibody competes for binding to human Axl with an Axlbinding domain of an antibody comprising the 10C9 VH domain (SEQ ID NO.3) and the 10C9 VL domain (SEQ ID NO. 4).

Preferably the antibody binds to the epitope bound by the antibodyobtainable from the hybridoma UT-10C9-B9, as described herein.

Preferably the antibody inhibits the binding of Axl to its ligand Gas6.Even more preferably, the antibody also downregulates Axl expression,inhibits Axl receptor signalling, and/or inhibits tumour growth.

According to a further aspect of the invention, there are providedvariants of the VH and VL domains of which the sequences are set outherein and that can be employed in antibodies for Axl and can beobtained by means of methods of sequence alteration or mutation andscreening. Such methods are also provided by the present invention.

Variable domain amino acid sequence variants of any of the VH and VLdomains whose sequences are specifically disclosed herein may beemployed in accordance with the present invention, as discussed.Particular variants may include one or more amino acid sequencealterations (addition, deletion, substitution and/or insertion of anamino acid residue), maybe less than about 20 alterations, less thanabout 15 alterations, less than about 10 alterations or less than about5 alterations, 4, 3, 2 or 1. Alterations may be made in one or moreframework regions and/or one or more CDR's.

An antibody according to the invention may be one that competes forbinding to antigen with any antibody that both binds the antigen andcomprises an antibody VH and/or VL domain disclosed herein, or VH CDR3disclosed herein, or variant of any of these. Competition betweenantibody may be assayed easily in vitro, for example using ELISA and/orby tagging a specific reporter molecule to one antibody that can bedetected in the presence of other untagged antibody(s), to enableidentification of antibodies that bind the same epitope or anoverlapping epitope.

Accordingly, the present invention comprises a variant of any antibodyspecifically disclosed herein, wherein the variant comprises one or moreamino acid sequence alterations in one or more framework regions and/orone or more CDRs. For example, the variant antibody may comprise no morethan 4 sequence alterations in any one CDR, such as no more than 3, nomore than 2, no more than 1 sequence alterations, or no sequencealterations in any one CDR (such as CDR3 of the VH domain). The variantantibody may compete for binding to Axl (for example, human Axl) with anAxl binding domain of an antibody comprising the 10C9 VH domain (SEQ IDNO. 3) and the 10C9 VL domain (SEQ ID NO. 4).

Thus a further aspect of the present invention provides an antibodycomprising a human antibody antigen-binding site that competes with 10C9for binding to human Axl.

In one aspect the present invention provides the antibody obtainablefrom the hybridoma UT-10C9-B9, as described herein.

Various methods are available in the art for obtaining antibodiesagainst Axl and which may compete with 10C9 for binding to Axl.

In a further aspect, the present invention provides a method ofobtaining one or more antibodies able to bind the antigen, the methodincluding bringing into contact a library of antibodies according to theinvention and said antigen, and selecting one or more antibody membersof the library able to bind said antigen.

The library may be displayed on the surface of bacteriophage particles,each particle containing nucleic acid encoding the antibody VH variabledomain displayed on its surface, and optionally also a displayed VLdomain if present.

Following selection of antibodies able to bind the antigen and displayedon bacteriophage particles, nucleic acid may be taken from abacteriophage particle displaying a said selected antibody. Such nucleicacid may be used in subsequent production of an antibody or an antibodyVH variable domain (optionally an antibody VL variable domain) byexpression from nucleic acid with the sequence of nucleic acid takenfrom a bacteriophage particle displaying a said selected antibody.

An antibody VH variable domain with the amino acid sequence of anantibody VH variable domain of a said selected antibody may be providedin isolated form, as may an antibody comprising such a VH domain.

Ability to bind Axl may be further tested, also ability to compete with10C9 for binding to Axl.

Alternatively, to screen for antibodies that bind to the epitope on Axlbound by an antibody of interest (e.g, those which block binding of the10C9 or 10G5 antibody to Axl), a routine cross-blocking assay such asthat described in Antibodies. A Laboratory Manual. Cold Spring HarborLaboratory. Ed Harlow and David Lane (1988), can be performed.

An antibody according to the present invention may bind Axl with theaffinity of 10C9.

An antibody of the invention may bind to murine, rat, monkey, non-humanprimate and/or human Axl. Preferably, the antibody binds to human andmonkey Axl. In some embodiments the antibody specifically binds primateAxl. For example, the antibody may specifically bind human and monkeyAxl. In one embodiment the antibody specifically binds only human Axl.

The antibody may be a chimeric, humanised, or CDR-grafted anti-Axlantibody. For example, the antibody may be a chimeric human/mouseantibody.

Binding affinity and neutralisation potency of different antibodies canbe compared under appropriate conditions.

In addition to antibody sequences, an antibody according to the presentinvention may comprise other amino acids, e.g. forming a peptide orpolypeptide, such as a folded domain, or to impart to the moleculeanother functional characteristic in addition to ability to bindantigen.

Antibodies of the invention may carry a detectable label, or may beconjugated to a toxin (such as a cytotoxin), enzyme, or an organicmoiety (e.g. via a peptidyl bond or linker).

Those skilled in the art are aware of numerous approaches to chemicallyconjugating molecules to proteins. In one embodiment of the presentinvention, the antibody can be conjugated to a detectable, fluorescentlabel, e.g. fluorescein isothiocyanate (FITC), or to a reporter enzymesuch as horseradish peroxidase (HRP)

In a preferred embodiment, the antibody is conjugated to a cytotoxicdrug with a formation of the antibody-drug conjugate (ADC). When theantibody is for pharmaceutical use, the bond linking the antibody anddrug is preferably stable in circulation (for example, bloodcirculation) but labile once the conjugate is sequesteredintracellularty. Thus, the antibody conjugated as an immunoconjugate maybe used in a method of treatment of, for example, cancer.

In further aspects, the invention provides an isolated nucleic acid thatcomprises a sequence encoding an antibody, VH domain and/or VL domainaccording to the present invention, and methods of preparing anantibody, a VH domain and/or a VL domain of the invention, that compriseexpressing said nucleic acid under conditions to bring about productionof said antibody, VH domain and/or VL domain, and recovering it.

Antibodies according to the invention may be used in a method oftreatment or diagnosis of the human or animal body, such as a method oftreatment (which may include prophylactic treatment) of a disease ordisorder in a human patient that comprises administering to said patientan effective amount of an antibody of the invention, or a conjugate, ordrug-conjugate thereof. Conditions treatable in accordance with thepresent invention include those discussed elsewhere herein.

Antibodies according to the invention may be used in a method ofimaging, for example, to determine the presence or location of cells towhich the antibody binds.

In a further aspect, the present invention provides a diagnostic kitcomprising an antibody according to the invention and one or morereagents to determine binding of the antibody to the antigen.

A further aspect of the present invention provides nucleic acid,generally isolated, encoding an antibody VH variable domain (SEQ ID NO:3) and/or VL variable domain (SEQ ID NO: 4) disclosed herein. In someembodiments the VH encoding nucleic acid has the sequence set out in SEQID NO: 1. In some embodiments the VL encoding nucleic acid has thesequence set out in SEQ ID NO: 2.

Another aspect of the present invention provides nucleic acid, generallyisolated, encoding a VH CDR or VL CDR sequence disclosed herein,especially a VH CDR selected from SEQ ID NOs 5, 6, and 7 or a VL CDRselected from SEQ ID NOs 8, 9, or 10, most preferably 10C9 CDR3 (SEQ IDNO: 7).

A further aspect provides a host cell transformed with nucleic acid ofthe invention.

A yet further aspect provides a method of production of an antibody VHvariable domain, the method including causing expression from encodingnucleic acid. Such a method may comprise culturing host cells underconditions for production of said antibody VH variable domain.

Analogous methods for production of VL variable domains and antibodiescomprising a VH and/or VL domain are provided as further aspects of thepresent invention.

A method of production may comprise a step of isolation and/orpurification of the product.

A method of production may comprise formulating the product into acomposition including at least one additional component, such as apharmaceutically acceptable excipient.

These and other aspects of the invention are described in further detailbelow.

The 10G5 Antibody

In one aspect, the present invention provides an isolated antibody thatbinds Axl and that comprises the 10G5 VH domain (SEQ ID NO: 21) and/orthe 10G5 VL domain (SEQ ID NO: 22). Preferably the bound Axl is humanAxl. In an alternative non-preferred aspect, the present inventionprovides an isolated antibody that binds Axl and that comprises the10G5[Q1E] VH domain (SEQ ID NO: 45) and/or the 10G5 VL domain (SEQ IDNO: 22). Preferably the bound Axl is human Axl.

In a preferred embodiment, the 10G5 VH domain (SEQ ID NO: 21) is pairedwith the 10G5 VL domain (SEQ ID NO: 22), so that an antibody antigenbinding site is formed comprising both the 10G5 VH and VL domains. Inother embodiments, the 10G5 VH is paired with a VL domain other than the10G5 VL; light-chain promiscuity is well established in the art. Forexample, in some embodiments the 10G5 VH domain (SEQ ID NO: 21) ispaired with the 10C9 VL domain (SEQ ID NO: 4). In an alternativenon-preferred aspect, the 10G5[Q1E] VH domain (SEQ ID NO: 45) is pairedwith the 10G5 VL domain (SEQ ID NO: 22), so that an antibody antigenbinding site is formed comprising both the 10G5 VH and VL domains. Inother embodiments, the 10G5 VH is paired with a VL domain other than the10G5 VL; light-chain promiscuity is well established in the art. Forexample, in some embodiments the 10G5[Q1E] VH domain (SEQ ID NO: 45) ispaired with the 10C9 VL domain (SEQ ID NO: 4).

One or more CDR's may be taken from the 10G5 VH or VL domain andincorporated into a suitable framework. This is discussed further below.10G5 VH CDR's 1, 2 and 3 are shown in SEQ ID Nos 23, 24 and 25,respectively. 10G5 VL CDR's 1, 2 and 3 are shown in SEQ ID Nos 26, 27,and 28, respectively.

In one aspect of the invention there is provided an antibody that bindsAxl and that comprises:

-   -   an antibody VH domain selected from the group consisting of the        10G5 VH domain (SEQ ID NO.21), the 10G5[Q1E] VH domain (SEQ ID        NO: 45), and a VH domain comprising a VH CDR3 with the amino        acid sequence of SEQ ID NO.25 and optionally one or more VH        CDR's with an amino acid sequence selected from SEQ ID NO.24 and        SEQ ID NO.23; and/or    -   an antibody VL domain selected from the group consisting of the        10G5 VL domain (SEQ ID NO. 22) and a VL domain comprising one or        more VL CDR's with an amino acid sequence selected from SEQ ID        NO.26, SEQ ID NO.27 and SEQ ID NO.28.

For example, the antibody may comprise an antibody VH domain comprisingthe VH CDR's with the amino acid sequences of SEQ ID NO.23, SEQ ID NO.24and SEQ ID NO.25. The antibody may further comprise an antibody VLdomain comprising the VL CDR's with the amino acid sequences of SEQ IDNO.26, SEQ ID NO.27 and SEQ ID NO.28.

In some embodiments the antibody comprises: (i) an antibody VH domaincomprising the VH CDR's with the amino acid sequences of SEQ ID NO.23,SEQ ID NO.24 and SEQ ID NO.25, and (ii) an antibody VL domain comprisingthe VL CDR's with the amino acid sequences of SEQ ID NO.26, SEQ ID NO.27and SEQ ID NO.28.

The antibody may comprise the 10G5 VH domain (SEQ ID NO. 21) and,optionally, further comprise the 10G5 VL domain (SEQ ID NO. 22). Inalternative, non-preferred embodiments, the antibody may comprise the10G5[Q1E] VH domain (SEQ ID NO: 45), and, optionally, further comprisethe 10G5 VL domain (SEQ ID NO. 22).

Preferably the antibody competes for binding to human Axl with an Axlbinding domain of an antibody comprising the 10G5 VH domain (SEQ ID NO.21) and the 10G5 VL domain (SEQ ID NO. 22).

Preferably the antibody binds to the epitope bound by the antibodyobtainable from the hybridoma WR-10G5-E5, as described herein.

Preferably the antibody inhibits the binding of Axl to its ligand Gas6.Even more preferably, the antibody also downregulates Axl expression,inhibits Axl receptor signalling, and/or inhibits tumour growth.

According to a further aspect of the invention, there are providedvariants of the VH and VL domains of which the sequences are set outherein and which can be employed in antibodies for Axl and can beobtained by means of methods of sequence alteration or mutation andscreening. Such methods are also provided by the present invention.

Variable domain amino acid sequence variants of any of the VH and VLdomains whose sequences are specifically disclosed herein may beemployed in accordance with the present invention, as discussed.Particular variants may include one or more amino acid sequencealterations (addition, deletion, substitution and/or insertion of anamino acid residue), maybe less than about 20 alterations, less thanabout 15 alterations, less than about 10 alterations or less than about5 alterations, 4, 3, 2 or 1. Alterations may be made in one or moreframework regions and/or one or more CDR's.

An antibody according to the invention may be one that competes forbinding to antigen with any antibody that both binds the antigen andcomprises an antibody VH and/or VL domain disclosed herein, or VH CDR3disclosed herein, or variant of any of these. Competition betweenantibody may be assayed easily in vitro, for example using ELISA and/orby tagging a specific reporter molecule to one antibody that can bedetected in the presence of other untagged antibody(s), to enableidentification of antibodies that bind the same epitope or anoverlapping epitope.

Alternatively, to screen for antibodies that bind to the epitope on Axlbound by an antibody of interest (e.g, those that block binding of the10C9 or 10G5 antibody to Axl), a routine cross-blocking assay such asthat described in Antibodies. A Laboratory Manual. Cold Spring HarborLaboratory. Ed Harlow and David Lane (1988), can be performed.

Accordingly, the present invention comprises a variant of any antibodyspecifically disclosed herein, wherein the variant comprises one or moreamino acid sequence alterations in one or more framework regions and/orone or more CDRs. For example, the variant antibody may comprise no morethan 4 sequence alterations in any one CDR, such as no more than 3, nomore than 2, no more than 1 sequence alterations, or no sequencealterations in any one CDR (such as CDR3 of the VH domain). The variantantibody may compete for binding to Axl (for example, human Axl) with anAxl binding domain of an antibody comprising the 10G5 VH domain (SEQ IDNO. 21) and the 10G5 VL domain (SEQ ID NO. 22).

Thus a further aspect of the present invention provides an antibodycomprising a human antibody antigen-binding site that competes with 10G5for binding to human Axl.

In one aspect the present invention provides the antibody obtainablefrom the hybridoma WR-10G5-E5, as described herein.

Various methods are available in the art for obtaining antibodiesagainst Axl and that may compete with 10G5 for binding to Axl.

In a further aspect, the present invention provides a method ofobtaining one or more antibodies able to bind the antigen, the methodincluding bringing into contact a library of antibodies according to theinvention and said antigen, and selecting one or more antibody membersof the library able to bind said antigen.

The library may be displayed on the surface of bacteriophage particles,each particle containing nucleic acid encoding the antibody VH variabledomain displayed on its surface, and optionally also a displayed VLdomain if present.

Following selection of antibodies able to bind the antigen and displayedon bacteriophage particles, nucleic acid may be taken from abacteriophage particle displaying a said selected antibody. Such nucleicacid may be used in subsequent production of an antibody or an antibodyVH variable domain (optionally an antibody VL variable domain) byexpression from nucleic acid with the sequence of nucleic acid takenfrom a bacteriophage particle displaying a said selected antibody.

An antibody VH variable domain with the amino acid sequence of anantibody VH variable domain of a said selected antibody may be providedin isolated form, as may an antibody comprising such a VH domain.

Ability to bind Axl may be further tested, also ability to compete with10G5 for binding to Axl.

An antibody according to the present invention may bind Axl with theaffinity of 10G5.

An antibody of the invention may bind to murine, rat, monkey, non-humanprimate and/or to human Axl. Preferably, the antibody binds to human andmonkey Axl. In some embodiments the antibody specifically binds primateAxl. For example, the antibody may specifically bind human and monkeyAxl. In one embodiment the antibody specifically binds only human Axl.

The antibody may be a chimeric, humanised, or CDR-grafted anti-Axlantibody. For example, the antibody may be a chimeric human/mouseantibody.

Binding affinity and neutralisation potency of different antibodies canbe compared under appropriate conditions.

In addition to antibody sequences, an antibody according to the presentinvention may comprise other amino acids, e.g. forming a peptide orpolypeptide, such as a folded domain, or to impart to the moleculeanother functional characteristic in addition to ability to bindantigen.

Antibodies of the invention may carry a detectable label, or may beconjugated to a toxin (such as a cytotoxin), enzyme, or an organicmoiety (e.g. via a peptidyl bond or linker).

Those skilled in the art are aware of numerous approaches to chemicallyconjugating molecules to proteins. In one embodiment of the presentinvention, the antibody can be conjugated to a detectable, fluorescentlabel, e.g. fluorescein isothiocyanate (FITC), or to a reporter enzymesuch as horseradish peroxidase (HRP)

In a preferred embodiment, the antibody is conjugated to a cytotoxicdrug with a formation of the antibody-drug conjugate (ADC). When theantibody is for pharmaceutical use, the bond linking the antibody anddrug is preferably stable in circulation (for example, bloodcirculation) but labile once the conjugate is sequesteredintracellularty. Thus, the antibody conjugated as an immunoconjugate maybe used in a method of treatment of, for example, cancer.

In further aspects, the invention provides an isolated nucleic acid thatcomprises a sequence encoding an antibody, VH domain and/or VL domainaccording to the present invention, and methods of preparing anantibody, a VH domain and/or a VL domain of the invention, that compriseexpressing said nucleic acid under conditions to bring about productionof said antibody, VH domain and/or VL domain, and recovering it.

Antibodies according to the invention may be used in a method oftreatment or diagnosis of the human or animal body, such as a method oftreatment (that may include prophylactic treatment) of a disease ordisorder in a human patient that comprises administering to said patientan effective amount of an antibody of the invention, or a conjugate, ordrug-conjugate thereof. Conditions treatable in accordance with thepresent invention include those discussed elsewhere herein.

Antibodies according to the invention may be used in a method ofimaging, for example, to determine the presence or location of cells towhich the antibody binds.

In a further aspect, the present invention provides a diagnostic kitcomprising an antibody according to the invention and one or morereagents to determine binding of the antibody to the antigen.

A further aspect of the present invention provides nucleic acid,generally isolated, encoding an antibody VH variable domain (SEQ ID NO:21), an antibody VH variable domain (SEQ ID NO: 45), and/or VL variabledomain (SEQ ID NO: 22) disclosed herein. In some embodiments the VHencoding nucleic acid has the sequence set out in SEQ ID NO: 19. In someembodiments the VL encoding nucleic acid has the sequence set out in SEQID NO: 20.

Another aspect of the present invention provides nucleic acid, generallyisolated, encoding a VH CDR or VL CDR sequence disclosed herein,especially a VH CDR selected from SEQ ID NOs 23, 24, and 25 or a VL CDRselected from SEQ ID NOs 26, 27, or 28, most preferably 10G5 CDR3 (SEQID NO: 25).

A further aspect provides a host cell transformed with nucleic acid ofthe invention.

A yet further aspect provides a method of production of an antibody VHvariable domain, the method including causing expression from encodingnucleic acid. Such a method may comprise culturing host cells underconditions for production of said antibody VH variable domain.

Analogous methods for production of VL variable domains and antibodiescomprising a VH and/or VL domain are provided as further aspects of thepresent invention.

A method of production may comprise a step of isolation and/orpurification of the product.

A method of production may comprise formulating the product into acomposition including at least one additional component, such as apharmaceutically acceptable excipient.

These and other aspects of the invention are described in further detailbelow.

Properties of the 10C9 Antibody

High Affinity for Axl

The 10C9 antibody described herein binds to human Axl with highaffinity. As described in Examples 5 and 13, the murine 10C9 antibodywas determined to have a K_(D) of 0.18 nM whilst the chimeric versionwas determined to have a K_(D) of 0.10 nM.

Accordingly, the 10C9 antibodies and variant thereof that are describedherein bind Axl with high affinity; preferably human Axl is bound withhigh affinity. In some embodiments, an antibody binds to Axl (or humanAxl) with a K_(D) no greater than 10⁻⁶ M, such as no greater than 5×10⁻⁷M, no greater than 10⁻⁷ M, no greater than 5×10⁻⁸ M, no greater than10⁻⁸ M, no greater than 5×10⁻⁹ M, no greater than 10⁻⁹ M, no greaterthan 5×10⁻¹⁰ M, no greater than 2×10⁻¹⁰ M, no greater than 1.1×10⁻¹⁰ M,no greater than 10⁻¹⁰ M, no greater than 5×10⁻¹¹ M, no greater than10⁻¹¹ M, no greater than 5×10⁻¹² M, no greater than 6×10⁻¹² M, nogreater than 10⁻¹² M, no greater than 5×10⁻¹³ M, no greater than 10⁻¹³M, no greater than 5×10⁻¹⁴ M, no greater than 10⁻¹⁴ M, no greater than5×10⁻¹⁵ M, or no greater than 10⁻¹⁵ M.

In some embodiments, an antibody binds to Axl (or human Axl) with aK_(D) from 10⁻⁸M to 10⁻¹⁰ M, from 10⁻¹⁰ M to 10⁻¹², from 10⁻¹² M to10⁻¹⁴, or from 10⁻¹⁴ M to 10⁻¹⁶.

The K_(D) may be determined and calculated as set out in Example 5, orExample 13.

The 10C9 antibody described herein is characterized by having a veryfast association rate (k_(on)). Specifically, in Example 5 the murine10C9 antibodies was determined to have the very fast association rate ofk_(on)=1.61×10⁶ M⁻¹s⁻¹, whilst in Example 13 the chimeric 10C9antibodies was determined to have the even faster association rate ofk_(on)=2.16×10⁶ M⁻¹s⁻¹. Accordingly, the antibodies described hereinpreferably bind human Axl with a fast association rate. In someembodiments, an antibody binds to Axl (or human Axl) with a k_(on) nolower than 10⁴ M⁻¹s⁻¹, such as no lower than 5×10⁴ M⁻¹s⁻¹, no lower than10⁵ M⁻¹s⁻¹, no lower than 5×10⁵ M⁻¹s⁻¹, no lower than 10⁶ M⁻¹s⁻¹, nolower than 1.5×10⁶ M⁻¹s⁻¹, no lower than 2×10⁶ M⁻¹s⁻¹, no lower than5×10⁶ M⁻¹s⁻¹, no lower than 10⁷ M⁻¹ s⁻¹, no lower than 2×10⁷ M⁻¹s⁻¹, nolower than 5×10⁷ M⁻¹s⁻¹, no lower than 10⁸ M⁻¹s⁻¹, no lower than 5×10⁸M⁻¹s⁻¹, or no lower than 10⁸ M⁻¹s⁻¹.

Properties of the 10G5 Antibody

High Affinity for Axl

The 10G5 antibody described herein binds to human Axl with highaffinity. As described in Examples 5 and 13, the murine 10G5 antibodywas determined to have a K_(D) of 0.53 nM whilst the chimeric versionwas determined to have a K_(D) of 0.10 nM.

Accordingly, the 10G5 antibodies and variant thereof that are describedherein bind Axl with high affinity; preferably human Axl is bound withhigh affinity. In some embodiments, an antibody binds to Axl (or humanAxl) with a K_(D) no greater than 10⁻⁶ M, such as no greater than 5×10⁻⁷M, no greater than 10⁻⁷ M, no greater than 5×10⁻⁸ M, no greater than10⁻⁸ M, no greater than 5×10⁻⁹ M, no greater than 10⁻⁹ M, no greaterthan 6×10⁻¹⁰ M, no greater than 5×10⁻¹⁰ M, no greater than 1.1×10⁻¹⁰ M,no greater than 10⁻¹⁰ M, no greater than 5×10⁻¹¹ M, no greater than10⁻¹¹ M, no greater than 5×10⁻¹² M, no greater than 6×10⁻¹² M, nogreater than 10⁻¹² M, no greater than 5×10⁻¹³ M, no greater than 10⁻¹³M, no greater than 5×10⁻¹⁴ M, no greater than 10⁻¹⁴ M, no greater than5×10⁻¹⁵ M, or no greater than 10⁻¹⁵ M.

In some embodiments, an antibody binds to Axl (or human Axl) with aK_(D) from 10⁻⁸ M to 10⁻¹⁰M, from 10⁻¹⁰ M to 10⁻¹², from 10⁻¹² M to10⁻¹⁴, or from 10⁻¹⁴ M to 10⁻¹⁶.

The K_(D) may be determined and calculated as set out in Example 5, orExample 13.

The 10G5 antibody described herein is characterized by having a veryfast association rate (k_(on)). Specifically, in Example 5 the murine10G5 antibodies was determined to have the very fast association rate ofk_(on)=0.83×10⁶ M⁻¹s⁻¹, whilst in Example 13 the chimeric 10C9antibodies was determined to have the even faster association rate ofk_(on)=1.64×10⁶ M⁻¹s⁻¹. Accordingly, the antibodies described hereinpreferably bind human Axl with a fast association rate. In someembodiments, an antibody binds to Axl (or human Axl) with a k_(on) nolower than 10⁴ M⁻¹s⁻¹, such as no lower than 5×10⁴ M⁻¹s⁻¹, no lower than10⁵ M⁻¹s⁻¹, no lower than 5×10⁵ M⁻¹s⁻¹, no lower than 10⁶ M⁻¹s⁻¹, nolower than 1.5×10⁶ M⁻¹s⁻¹, no lower than 2×10⁶ M⁻¹s⁻¹, no lower than5×10⁶ M⁻¹s⁻¹, no lower than 10⁷ M⁻¹s⁻¹¹, no lower than 2×10⁷ M⁻¹s⁻¹, nolower than 5×10⁷ M⁻¹s⁻¹, no lower than 10⁸ M⁻¹s⁻¹, no lower than 5×10⁸M⁻¹s⁻¹, or no lower than 10⁸ M⁻¹s⁻¹.

Properties of Both the 10C9 & 10G5 Antibodies

Specific Binding

Generally, the terms ‘specific’ and ‘specifically binds’ may be used torefer to the situation in which an antibody will not show anysignificant binding to molecules other than its specific bindingpartner(s). For example, an antibody that ‘specifically binds’ human Axlwould not show any significant binding for murine Axl.

The term is also applicable where e.g. an antibody is specific for aparticular epitope that is carried by a number of antigens, in whichcase an antibody that ‘specifically binds’ an epitope will be able tobind to all of the various antigens that carry the recognised epitope.

Typically, specificity may be determined by means of a binding assaysuch as ELISA employing a panel of antigens.

The 10C9 and 10G5 antibodies described herein bind to human Axl withhigh specificity. That is, the 10C9 and 10G5 antibodies ‘specificallybind’ human Axl. This is demonstrated in the examples, where it is shownthat:

-   -   (1) In Example 2, 10C9 and 10G5 show no significant binding to        recombinant antigens derived from hMer and hTyro3, the other        members of the human TAM receptor tyrosine kinase family;    -   (2) In Example 3, 10C9 and 10G5 bind strongly to human Axl, but        show no binding to murine Axl (this is in contrast to murine Axl        ligand, murine Gas 6, that binds strongly to both murine and        human Axl, as well as (more weakly) binding human Tyro3);    -   (3) In Example 4, 10C9 and 10G5 bind strongly to Axl from the        cynomolgus monkey (Macaca fascicularis).

Accordingly, the antibodies described herein preferably specificallybind primate Axl. In some embodiments the antibodies described hereinspecifically bind human and monkey (such as Macaca fascicularis) Axl. Inone embodiment the antibodies specifically bind only human Axl.

In some embodiments of the present invention, the antibodies describedherein show no significant binding to human Tyro3 and/or human Mer. Insome embodiments the antibodies described herein show no significantbinding to murine Axl. In some embodiments the antibodies describedherein show no significant binding to any of human Tyro3, human Mer, ormurine Axl.

Whether an antibody shows “no significant binding” to an antigen can bereadily determined by the skilled person using, for example, thetechniques described in Examples 2 and 3. In some embodiments, anantibody is deemed to show “no significant binding” to a particularantigen if it binds the antigen with a K_(D) greater than 10⁻³ M, suchas greater than 10⁻² M, greater than 10⁻¹ M, or greater than 1 M. TheK_(D) may be determined and calculated as set out in Example 5.

Inhibition of Axl/Gas6 Binding

The 10C9 and 10G5 antibodies described herein inhibit the binding of Axlto its ligand Gas6.

FIG. 5 shows the results of the competitive binding assay described inExample 6. The results show that immobilised rhAxl saturated with 10C9cannot be bound by subsequently added 10C9, or any of 10G5, rhGas6 (aknown ligand of rhAxl), or rmGas6. This indicates that the areas of theAxl molecule bound by 10C9, 10G5 and Gas6 are in close proximity to oneanother. In contrast, the binding of 10C9 did no inhibit the binding ofthe MAB154 anti-Axl antibody, indicating that 10C9 and MAB154 bind todistinct parts of the Axl molecule.

Accordingly, in preferred embodiments the antibodies described hereininhibit the binding of Axl to Gas6 (for example, rhAxl to rhGas6). Thatis, preferably the antibodies described herein compete with human Gas 6for binding to human Axl. Most preferably, inhibition of Axl/Gas 6binding is such that no significant binding of Gas6 can be observed toan Axl sample saturated with the antibody (for example, no more than 1%of the binding observed to an Axl sample that has not been previouslyexposed to the antibody). Inhibition of Gas 6 binding may be assessedusing the competitive binding assay described in Example 6.

Inhibition of Axl Receptor Expression

The antibodies of the invention lead to a significant reduction in theexpression of Axl.

FIG. 8 shows the results of the Western Blot analyses described inExample 9, in which MBA-MD-231 cells are incubated overnight with one ofa range of antibodies and then tested for Axl expression. The resultsshow that incubation with 10C9 leads to a significant reduction in theamount of Axl receptor protein present in the cell, indicating thatbinding of the 10C9 antibody downregulates the expression of the Axlreceptor.

Accordingly, in preferred embodiments the antibodies of the inventiondownregulate expression of the Axl receptor.

In some embodiments, the antibody of the invention reduces Axl receptorexpression to less than 80% of the level observed in an otherwiseidentically treated sample that is not contacted with to the antibody.In some embodiments, the antibody of the invention reduces Axl receptorexpression to less than 70%, less than 60%, less than 50%, less than40%, less than 30%, less than 20%, or less than 10% of the levelobserved in an otherwise identically treated sample that is notcontacted with the antibody. The level of Axl receptor expression may beassessed using the assay described in Example 9; a number of methods foraccurately quantifying bands on Western blots are known in the art—see,for example, Taylor et al. Mol Biotechnol. 2013; 55(3): 217-226.

In some embodiments down-regulation of Axl receptor expression occursrapidly; for example, in some embodiments a reduction of Axl receptorexpression to less than 80% of the level observed in an otherwiseidentically treated sample that is not contacted with the antibody isobserved within 12 hours of contacting the sample with the antibody, forexample within 12 hours, within 6 hours, within 3 hours, or within 1hour of contacting the sample with the antibody.

In some embodiments, the antibody causes persistent downregulation ofAxl receptor expression. For example, in some embodiments the level ofAxl receptor expression in a sample contacted with the antibody remainsbelow 50% of the level observed in an otherwise identically treatedsample that is not contacted with the antibody for at least 6 hoursfollowing contacting the sample with the antibody, such as at least 12hours, at least 24 hours, at least 48 hours, or at least 96 hours.

Without wishing to be bound by theory, it is believed the observeddown-regulation of Axl expression is caused by the antibody/Axl receptorcomplex being internalised and degraded by the cell. Internalisation ofthe antibody is highly advantageous for applications where it isdesirable to get the antibody, or a molecule linked to the antibody,into a target cell. For example, where the target is a cancerous celland the antibody is linked to a cytotoxic drug.

Accordingly, in preferred embodiments the antibodies of the inventionincreases the rate of Axl receptor internalization.

In some embodiments, the antibody of the invention increases the rate ofAxl receptor internalization to at least 110% of the level observed inan otherwise identically treated sample that is not contacted with theantibody. In some embodiments, the antibody of the invention increasesthe rate of Axl receptor internalization to at least 120%, at least130%, at least 140%, at least 150%, at least 160%, at least 170%, atleast 180%, at least 190%, at least 200%, at least 500%, at least 1000%of the level observed in an otherwise identically treated sample that isnot contacted with the antibody.

The level of Axl receptor internalization may be assessed using any oneof the receptor internalisation assay known in the art; for example, themethod described in Koenig et al. Methods in Molecular Biology Volume259, 2004, pp 249-273.

Inhibition of Axl Receptor Signalling

Consistent with the observations that the antibodies of the invention(1) inhibit the binding of the Axl receptor to natural ligands such asGas6, and (2) downregulate the expression of the Axl receptor, theantibodies of the invention inhibit ligand-induced signalling downstreamof the Axl receptor. This is demonstrated in FIG. 9 , where it can beseen that the presence of the 10C9 antibody significantly reduces thedegree to which Aid's Serine 473 is phosphorylated on addition of theAxl ligand Gas6.

Accordingly, in preferred embodiments the antibodies of the inventioninhibit Axl activity. The inhibited activity may be constitutive Axlactivity.

In some embodiments the antibodies of the invention inhibit Axldownstream signalling, for example the phosphorylation of Akt at Serine473. In some embodiments, the phosphorylation of Aid at Serine 473 in asample contacted with the antibody of the invention is less than 80% ofthe level observed in an otherwise identically treated sample that isnot contacted with the antibody. In some embodiments, thephosphorylation of Aid at Serine 473 in a sample contacted with theantibody of the invention is less than 70%, less than 60%, less than50%, less than 40%, less than 30%, less than 20%, or less than 10% ofthe level observed in an otherwise identically treated sample that isnot contacted with the antibody. The level of phosphorylation of Aid atSerine 473 may be assessed using the assay described in Example 10; anumber of methods for accurately quantifying bands on Western blots areknown in the art—see, for example, Taylor et al. Mol Biotechnol. 2013;55(3): 217-226.

By virtue of inhibiting Axl receptor signalling, the antibodies of theinvention are also expected to influence a range of processes in whichAxl-receptor signalling plays a role.

For example, it is known that Axl-receptor signalling stimulates Gas6dependant cell proliferation and inhibits cell-death, thus supportingtumour growth. It is also known that Axl-receptor signalling stimulatesEpithelial-Mesenchymal transition (EMT) and thus promotes tumourmetastases.

Accordingly, in some embodiments, the antibodies of the inventionpromotes cell death, for example by apoptosis. Preferably the cell is atumour cell, such as a circulating tumour cell or a metastatic cell. Forexample, in some embodiments, the antibody of the invention increasesthe rate of cell-death to at least 110% of the level observed in anotherwise identically treated sample that is not contacted with theantibody. In some embodiments, the antibody of the invention increasesthe rate of cell death to at least 120%, at least 130%, at least 140%,at least 150%, at least 160%, at least 170%, at least 180%, at least190%, at least 200%, at least 500%, at least 1000% of the level observedin an otherwise identically treated sample that is not exposed to theantibody. The rate of ell death may be measured by, for example BrdUincorporation assay, MTT, [³H]-thymidine incorporation (e.g., TopCountassay (PerkinElmer)), cell viability assays (e.g., CellTiter-Glo(Promega)), DNA fragmentation assays, caspase activation assays, tryptanblue exclusion, chromatin morphology assays and the like.

In some embodiments, the antibodies of the invention inhibit Axldownstream signalling. In some embodiments, the antibodies of theinvention inhibit Gas6 dependent cell proliferation.

In some embodiments, the antibodies of the invention inhibitinflammatory cytokine expression from tumour-associated macrophages.

Inhibition of Tumour Growth

Consistent with the role of Axl and the EMT pathway in tumour growth,the antibodies of the invention reduce the rate of growth of bothhaematological and non-haematological tumours; this is demonstrated bythe data shown in FIGS. 14 and 15 , as obtained through the methodsdescribed in Examples 14 and 15.

Accordingly, in preferred embodiments the antibodies of the inventioninhibit tumour growth and/or metastasis by, for example, modulatingtumour stromal function.

In some embodiments the antibodies of the invention inhibit tumourgrowth by at least 10% compared to a control tumour. That is, the volumeof the antibody treated tumour is no more than 90% of the volume of thecontrol tumour. For example, in some embodiments the antibodies of theinvention inhibit tumour growth by at least 20% compared to a controltumour, such as at least 30%, at least 40%, at least 50%, at least 60%,at least 70%, at least 80%, or at least 90%.

In some embodiments, the effect of the antibody on tumour growth isassayed as described in example 14. In some embodiments, the effect ofthe antibody on tumour growth is assayed as described in example 15.

Definitions

Antibody

This term describes an immunoglobulin whether natural or partly orwholly synthetically produced. The term also covers any polypeptide orprotein comprising an antibody antigen-binding domain. Antibodyfragments that comprise an antibody antigen-binding domain include wholeantibodies (for example an IgG antibody comprising VH, CH1, CH2, CH3,VL, and CL domains in the canonical arrangement), or fragments of wholeantibodies that retain their binding activity for a target antigen. Suchfragments include Fv (fragment variable), Fab (fragment antibodybinding) and F(ab′)₂ fragments, as well as single-chain Fv antibodies(scFv), dsFv, minibodies, diabodies, single-chain diabodies, tandemscFv, TandAb, bi-body, tri-body, kappa(lambda) body, BiTE, DVD-Ig, SIP,SMIP, or DART. Furthermore, the antibodies and fragments thereof may behumanised antibodies, for example as described in EP239400A. Forexample: monoclonal and polyclonal antibodies, recombinant antibodies,proteolytic and recombinant fragments of antibodies (Fab, Fv, scFv,diabodies), single-domain antibodies (VHH, sdAb, nanobodies, IgNAR,VNAR), and proteins unrelated to antibodies, that have been engineeredto have antibody-like specific binding (antibody mimetics), such as thefollowing, but not limited to:

Name Based on: Adnectins/Monobodies 10th type III domain of humanfibronectin (10Fn3), 10 kDa Affibodies Protein A, Z domain, 6 kDa)Affilins Human γ-crystallin/human ubiquitin (10-20 kDa) Affitins Sac7d(from Sulfolobus acidocaldarius), 7 kDa Anticalins Lipocalins, 20 kDaAvimers Domains of various membrane receptors, 9-18 kDa DARPins Ankyrinrepeat motif, 14 kDa Evibody Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4),15 kDa Fynomers Fyn, SH3 domain, 7 kDa Kunitz domain Various proteaseinhibitors, 6 kDa peptides

An antibody may comprise all or apportion of an antibody heavy chainconstant region and/or an antibody light chain constant region.

It is possible to take monoclonal and other antibodies and usetechniques of recombinant DNA technology to produce engineeredantibodies or chimeric molecules, that retain the specificity of theoriginal antibody. Such techniques may involve ligation of DNA fragmentsencoding the immunoglobulin variable regions, or the complementaritydetermining regions (CDRs), of an antibody with genes coding for theimmunoglobulin constant regions, or the constant regions plus frameworkregions, of a different immunoglobulin. See, for instance, EP-A-184187,GB 2188638A or EP-A-239400. A hybridoma or other cell producing anantibody may be subject to genetic mutation or other changes, that mayor may not alter the binding specificity of antibodies produced.

As antibodies can be modified in a number of ways, the term “antibodymolecule” should be construed as covering any polypeptide or othermolecule having an antibody-derived antigen-binding domain with therequired specificity. Thus, this term covers antibody fragments andderivatives, including any polypeptide comprising an immunoglobulinbinding domain, whether natural or wholly or partially synthetic.Chimeric molecules comprising an immunoglobulin binding domain, orequivalent, fused to another polypeptide are therefore included. Cloningand expression of chimeric antibodies are described in EP-A-0120694 andEP-A-0125023.

It has been shown that fragments of a whole antibody can perform thefunction of binding antigens. Examples of binding fragments are (i) theFab fragment consisting of VL, VH, CL and CH1 domains; (ii) the Fdfragment consisting of the VH and CH1 domains; (iii) the Fv fragmentconsisting of the VL and VH domains of a single antibody; (iv) the dAbfragment (Ward, E. S. et al., Nature 341, 544-546 (1989)) that consistsof a VH domain; (v) isolated CDR regions; (vi) F(ab′)2 fragments, abivalent fragment comprising two linked Fab fragments; (vii) singlechain Fv molecules (scFv), wherein a VH domain and a VL domain arelinked by a peptide linker that allows the two domains to associate toform an antigen binding site (Bird et al, Science, 242, 423-426, 1988;Huston et al, PNAS USA, 85, 5879-5883, 1988); (viii) bispecific singlechain Fv dimers (PCT/US92/09965) and (ix) “diabodies”, multivalent ormultispecific fragments constructed by gene fusion (WO94/13804; P.Holliger at al, Proc. Natl. Acad. Sci. USA 90, 6444-6448, 1993). Fv,scFv or diabody molecules may be stabilised by the incorporation ofdisulphide bridges linking the VH and VL domains (Y. Reiter et al,Nature Biotech, 14, 1239-1245, 1996). Minibodies comprising a scFvjoined to a CH3 domain may also be made (S. Hu et al, Cancer Res., 56,3055-3061, 1996).

The antibody may be bispecific or multispecific. Where bispecificantibodies are to be used, these may be conventional bispecificantibodies, that can be manufactured in a variety of ways (Holliger, P.and Winter G. Current Opinion Biotechnol. 4, 446-449 (1993)), e.g.prepared chemically or from hybrid hybridomas, or may be any of thebispecific antibody fragments mentioned above. Diabodies and scFv can beconstructed without an Fc region, using only variable domains,potentially reducing the side effects, such as those due to the antibodyeffector functions, or human-anti-mouse antibody (HAMA) response in caseof using antibodies of murine origin.

Bispecific diabodies, as opposed to bispecific whole antibodies, mayalso be particularly useful because they can be readily constructed andexpressed in bacteria (e.g. Escherichia coli). Diabodies (and many otherpolypeptides such as antibody fragments) of appropriate bindingspecificities can be readily selected using phage display (WO94/13804)from the antibody libraries. If one arm of the diabody is to be keptconstant, for instance, with a specificity directed against Axl, then alibrary can be made where the other arm is varied and an antibody ofappropriate specificity selected. Bispecific whole antibodies may bemade by “knobs-into-holes” engineering (J. B. B. Ridgeway et al, ProteinEng., 9, 616-621, 1996).

Sample

As used herein, a “sample” may be a single cell or a population ofcells. The cell(s) may be normal, healthy cell(s) or may be tumourcells, such as circulating tumour cells.

The sample may be in vivo, ex vivo, or in vitro. For example, the samplemay be an in vivo tumour mass, or an in vitro cell population.

Antigen Binding Domain

This describes the part of an antibody molecule that comprises the areathat recognizes and specifically binds to and is complementary part orall of an antigen. Where an antigen is large, an antibody may only bindto a particular part of the antigen, which part is termed an epitope. Anantigen binding domain may be provided by one or more antibody variabledomains (e.g. a so-called Fd antibody fragment consisting of a VHdomain). Preferably, an antigen binding domain comprises an antibodylight chain variable region (VL) and an antibody heavy chain variableregion (VH).

Specific Proteins

Human Axl

As used herein, ‘human Axl’ refers to the Axl member of the human TAMfamily of receptor tyrosine kinases. Human Axl occurs in the followingisoforms:

Axl Isoform mRNA: NCBI reference Polypeptide: NCBI reference ANM_001278599.1, GI: 520260398, NP_001265528.1, GI: 520260399, recordupdate date: Nov 28, 2014 record update date: Nov 28, 2014 12:30 AM12:30 AM (SEQ ID NO. 37) B NM_001699.5,_GI: 520260376, NP_001690.2,_GI:21536468, record update date: Nov 28, 2014 record update date: Nov 28,2014 12:30 AM 12:30 AM C NM_021913.4,_GI: 520260356, recordNP_068713.2,_GI: 21536466, update date: Nov 28, 2014 12:30 AM recordupdate date: Nov 28, 2014 12:30 AM

In some embodiments, the human Axl polypeptide corresponds to Isoform“A”, shown above.

In some embodiments, the human Axl polypeptide corresponds to Isoform“B”, shown above.

In some embodiments, the human Axl polypeptide corresponds to Isoform“C”, shown above.

Murine Axl

As used herein, ‘murine Axl’ refers to the Axl member of the murine TAMfamily of receptor tyrosine kinases. Murine Axl occurs in the followingisoforms:

Axl Isoform mRNA: NCBI reference Polypeptide: NCBI reference ANM_001190974.1, GI: 300794859, NP_001177903.1, GI: 300794860, recordupdate date: Sep 5, 2014 record update date: Sep 5, 2014 08:46 PM 08:46PM (SEQ ID NO. 38) B NM_001190975.1, GI: 300794883, NP_001177904.1, GI:300794884, record update date: Sep 5, 2014 record update date: Sep 5,2014 08:46 PM 08:46 PM C NM_009465.4, GI: 300794836, record NP_033491.2,GI: 31542164, update date: Sep 5, 2014 08:46 PM record update date: Sep5, 2014 08:46 PM

In some embodiments, the murine Axl polypeptide corresponds to Isoform“A”, shown above.

In some embodiments, the murine Axl polypeptide corresponds to Isoform“B”, shown above.

In some embodiments, the murine Axl polypeptide corresponds to Isoform“C”, shown above.

Human Tyro3

As used herein, ‘human Tyro3’ refers to the Tyro3 member of the humanTAM family of receptor tyrosine kinases. In some embodiments, the humanTyro3 polypeptide corresponds to NCBI accession no. NP_006284.2,GI:27597078, record update date: Nov. 28, 2014 12:30 AM (SEQ ID NO.39).In one embodiment, the nucleic acid encoding the human Tyro3 polypeptidecorresponds to NCBI accession no. NM_006293.3, GI:295842183, recordupdate date: Nov. 28, 2014 12:30 AM.

Human Mer

As used herein, ‘human Mer’ refers to the Mer member of the human TAMfamily of receptor tyrosine kinases. In some embodiments, the human Merpolypeptide corresponds to NCBI accession no. NP_006334.2, GI:66932918,record update date: Sep. 6, 2014 04:03 AM (SEQ ID NO.40). In oneembodiment, the nucleic acid encoding the human Mer polypeptidecorresponds to NCBI accession no. NM_006343, version no. NM_006343.2GI:66932917, record update date: Sep. 6, 2014 04:03 AM.

Human Akt3

As used herein, ‘human Atk3’ refers to the Atk3 member of the human AKTsubfamily of serine/threonine protein kinases. Human Akt3 occurs in thefollowing isoforms:

Akt 3 Isoform mRNA: NCBI reference Polypeptide: NCBI reference ANM_001206729.1, GI: 332078558, NP_001193658.1, GI: 332078559, recordupdate date: Sep 6, 2014 record update date: Sep 6, 2014 02:43 AM 02:43AM (SEQ ID NO. 41) B NM_005465.4, GI: 332078467, record NP_005456.1, GI:4885549, record update date: Sep 6, 2014 02:43 AM update date: Sep 6,2014 02:43 AM C NM_181690.2, GI: 332078557, record NP_859029.1, GI:32307163, update date: Sep 6, 2014 02:43 AM record update date: Sep 6,2014 02:43 AM

In some embodiments, the human Aid polypeptide corresponds to Isoform“A”, shown above. In some embodiments, the human Aid polypeptidecorresponds to Isoform “B”, shown above. In some embodiments, the humanAid polypeptide corresponds to Isoform “C”, shown above.

Human Gas6

As used herein, ‘human Gas6’ (Growth Arrest Specific 6) refers to aligand of the TAM family of receptor tyrosine kinases. In someembodiments, the human Gas6 polypeptide corresponds to NCBI accessionno. NP_000811.1, GI:4557617, record update date: Sep. 6, 2014 02:44 AM(SEQ ID NO.42). In one embodiment, the nucleic acid encoding the humanGas6 polypeptide corresponds to NCBI accession no. NM_000820.3,GI:673038877, record update date: Sep. 6, 2014 02:44 AM.

BSA

As used herein, ‘BSA’ refers to Bovine Serum Albumin. In someembodiments BSA corresponds to ‘A9647-Bovine Serum Albumin’ (SigmaAldrich). In some embodiments BSA corresponds to Genbank accession no.CAA76847, version no. CAA76847.1 GI:3336842, record update date: Jan. 7,2011 02:30 PM.

Comprise

This is generally used in the sense of “include”, that is to saypermitting the presence of one or more features or components.

Isolated

This refers to the state in which antibodies of the invention, ornucleic acid encoding such antibody, will generally be in accordancewith the present invention. Antibody and nucleic acid will be free orsubstantially free of material with which they are naturally associatedsuch as other polypeptides or nucleic acids with which they are found intheir natural environment, or the environment in which they are prepared(e.g. cell culture) when such preparation is by recombinant DNAtechnology practiced in vitro or in vivo. Antibodies and nucleic acidmay be formulated with diluents or adjuvants and still for practicalpurposes be isolated—for example the antibody will normally be mixedwith gelatin or other carriers if used to coat microtitre plates for usein immunoassays, or will be mixed with pharmaceutically acceptablecarriers or diluents when used in diagnosis or therapy. Antibodies maybe glycosylated, either naturally or by systems of heterologouseukaryotic cells (e.g. CHO or NS0 (ECACC 85110503) cells), or they maybe (for example, if produced by expression in a prokaryotic cell)non-glycosylated.

Substantially as Set Out

By “substantially as set out” it is meant that the relevant CDR or VH orVL domain of the invention will be either identical or highly similar tothe specified regions of which the sequence is set out herein. By“highly similar” it is contemplated that from 1 to 5, preferably from 1to 4 such as 1 to 3 or 1 or 2, or 3 or 4, amino acid substitutions maybe made in the CDR and/or VH or VL domain.

Frameworks Supporting CDRs

The structure for carrying a CDR of the invention will generally be ofan antibody heavy or light chain sequence or substantial portion thereofin which the CDR is located at a location corresponding to the CDR ofnaturally occurring VH and VL antibody variable domains encoded byrearranged immunoglobulin genes. The structures and locations ofimmunoglobulin variable domains may be determined by reference to(Kabat, E. A. et al, Sequences of Proteins of Immunological Interest.4th Edition. US Department of Health and Human Services. 1987, andupdates thereof, now available on the Internet (immuno.bme.nwu.edu orfind “Kabat” using any search engine).

Variable domains employed in the invention may be obtained from anygerm-line or rearranged mouse or human variable domain, or may be asynthetic variable domain based on consensus sequences of known mouse orhuman variable domains. A CDR sequence of the invention (e.g. CDR3) maybe introduced into a repertoire of variable domains lacking a CDR (e.g.CDR3), using recombinant DNA technology.

For example, Marks et al (Bio/Technology, 1992, 10:779-783) describemethods of producing repertoires of antibody variable domains in whichconsensus primers directed at or adjacent to the 5′-end of the variabledomain area are used in conjunction with consensus primers to the thirdframework region of human VH genes to provide a repertoire of VHvariable domains lacking a CDR3. Marks et al. further describe how thisrepertoire may be combined with a CDR3 of a particular antibody. Usinganalogous techniques, the CDR3-derived sequences of the presentinvention may be shuffled with repertoires of VH or VL domains lacking aCDR3, and the shuffled complete VH or VL domains combined with a cognateVL or VH domain to provide antibodies of the invention. The repertoiremay then be displayed in a suitable host system such as the phagedisplay system of WO92/01047 so that suitable antibodies may beselected. A repertoire may consist of from anything from 10⁴ individualantibody upwards, for example from 10⁶ to 10⁸ or 10¹⁰ antibodies.

Analogous shuffling or combinatorial techniques are also disclosed byStemmer (Nature, 1994, 370:389-391), who describes the technique of DNAshuffling in relation to a 3-lactamase gene but observes that theapproach may be used for the generation of antibodies.

A further alternative is to generate novel VH or VL regions carrying aCDR-derived sequences of the invention using random mutagenesis of oneor more selected VH and/or VL genes to generate mutations within theentire variable domain. Such a technique is described by Gram et al(1992, Proc. Natl. Acad. Sci., USA, 89:3576-3580), who used error-pronePCR.

Another method that may be used is to direct mutagenesis to CDR regionsof VH or VL genes. Such techniques are disclosed by Barbas et al. (1994,Proc. Natl. Acad. Sci., USA, 91:3809-3813) and Schier et al. (1996, J.Mol. Biol. 263:551-567).

All the above-described techniques are known as such in the art and inthemselves do not form part of the present invention. The skilled personwill be able to use such techniques to provide antibodies of theinvention using routine methodology in the art.

Epitope-Specific Antibodies

A further aspect of the invention provides a method for obtaining anantibody specific for an Axl epitope, the method comprising providing byway of addition, deletion, substitution or insertion of one or moreamino acids in the amino acid sequence of a VH domain set out herein aVH domain that is an amino acid sequence variant of the VH domain,optionally combining the VH domain thus provided with one or more VLdomains, and testing the VH domain or VH/VL combination or combinationsto identify a antibody or an antibody antigen binding domain specificfor Axl. Said VL domain may have an amino acid sequence which issubstantially as set out herein.

To screen for antibodies that bind to the epitope on Axl bound by anantibody of interest (e.g, those that block binding of the 10C9 or 10G5antibody to Axl), a routine cross-blocking assay such as that describedin Antibodies. A Laboratory Manual. Cold Spring Harbor Laboratory. EdHarlow and David Lane (1988), can be performed.

An analogous method may be employed in which one or more sequencevariants of a VL domain disclosed herein are combined with one or moreVH domains.

A further aspect of the invention provides a method of preparing anantibody specific for Axl, which method comprises:

-   -   (a) providing a starting repertoire of nucleic acids encoding a        VH domain that either includes a CDR3 to be replaced or lack a        CDR3 encoding region;    -   (b) combining said repertoire with a donor nucleic acid encoding        an amino acid sequence substantially as set out herein for a VH        CDR3 such that said donor nucleic acid is inserted into the CDR3        region in the repertoire, so as to provide a product repertoire        of nucleic acids encoding a VH domain;    -   (c) expressing the nucleic acids of said product repertoire;    -   (d) selecting an antibody specific for Axl; and    -   (e) recovering said antibody or nucleic acid encoding it.

Again, an analogous method may be employed in which a VL CDR3 of theinvention is combined with a repertoire of nucleic acids encoding a VLdomain that either includes a CDR3 to be replaced or lack a CDR3encoding region.

Similarly, one or more, or all three CDRs may be grafted into arepertoire of VH or VL domains that are then screened for an antibody orantibodies specific for Axl.

A substantial portion of an immunoglobulin variable domain will compriseat least the three CDR regions, together with their interveningframework regions. Preferably, the portion will also include at leastabout 50% of either or both of the first and fourth framework regions,the 50% being the C-terminal 50% of the first framework region and theN-terminal 50% of the fourth framework region. Additional residues atthe N-terminal or C-terminal end of the substantial part of the variabledomain may be those not normally associated with naturally occurringvariable domain regions. For example, construction of antibodies of thepresent invention made by recombinant DNA techniques may result in theintroduction of N- or C-terminal residues encoded by linkers introducedto facilitate cloning or other manipulation steps. Other manipulationsteps include the introduction of linkers to join variable domains ofthe invention to further protein sequences including immunoglobulinheavy chains, other variable domains (for example in the production ofdiabodies) or protein labels as discussed in more details below.

Although in a preferred aspect of the invention antibodies comprising apair of VH and VL domains are preferred, single binding domains based oneither VH or VL domain sequences form further aspects of the invention.It is known that single immunoglobulin domains, especially VH domains,are capable of binding target antigens in a specific manner.

In the case of either single binding domains, these domains may be usedto screen for complementary domains capable of forming a two-domainantibody able to bind Axl.

This may be achieved by phage display screening methods using theso-called hierarchical dual combinatorial approach as disclosed inWO92/01047 in which an individual colony containing either an H or Lchain clone is used to infect a complete library of clones encoding theother chain (L or H) and the resulting two-chain antibody is selected inaccordance with phage display techniques such as those described in thatreference. This technique is also disclosed in Marks et al., ibid.

Antibodies of the present invention may further comprise antibodyconstant regions or parts thereof. For example, an antibody of thepresent invention may comprise a CL, CH1, CH2, and/or a CH3 domain (orany combination thereof). A VL domain may be attached at its C-terminalend to antibody light chain constant domains including human Cκ or Cλchains, preferably Cκ chains. Similarly, an antibody based on a VHdomain may be attached at its C-terminal end to all or part of animmunoglobulin heavy chain derived from any antibody isotype, e.g. IgG,IgA, IgE and IgM and any of the isotype sub-classes. Fc regions such asΔnab and Δnac as disclosed in WO99/58572 may be employed.

Chimeric. Humanised and CDR-Grafted Antibodies

As used herein “chimeric” antibodies or “humanised” antibodies or“CDR-grafted” include any combination of the herein described anti-Axlantibodies, or any CDR derived therefrom combined with one or moreproteins or peptides derived from a non-murine, preferably, humanantibody.

Chimeric or humanised antibodies include those wherein the CDR's arederived from one or more of the herein described anti-Axl antibodies andat least a portion, or the remainder of the antibody is derived from oneor more human antibodies. Thus, the human part of the antibody mayinclude the frameworks, CL (e.g. Cκ or Cλ), CH domains (e.g., CH1, CH2,CH3), hinge regions that are substantially non-immunogenic in humans.

The regions of the antibody that are derived from human antibodies neednot have 100% identity with human antibodies. In a preferred embodiment,as few of the mouse amino acid residues as possible are retained inorder for the immunogenicity to be negligible, but the mouse residuesmay be retained as necessary to support the antigen binding site formedby the CDR's while simultaneously maximizing the humanization of theantibody. Such changes or variations optionally and preferably retain orreduce the immunogenicity in humans or other species relative tonon-modified antibodies.

It should be noted that a humanised antibody can be produced by anon-human animal or prokaryotic or eukaryotic cell that is capable ofexpressing functionally rearranged human immunoglobulin (e.g., heavychain and/or light chain) genes. Further, when the antibody is a singlechain antibody, it can comprise a linker peptide that is not found innative human antibodies. For example, an scFv can comprise a linkerpeptide, such as two to about twenty glycine or other amino acidresidues (preferably glycine and serine residues (e.g., Gly₄Ser orGly₂Ser repeats)), that connects the variable region of the heavy chainand the variable region of the light chain. Such linker peptides areconsidered to be non-immunogenic in humans. In some embodiments thelinker is of at least 12 amino acids in length.

Antibody humanisation can be performed by, for example, synthesizing acombinatorial library comprising all six CDRs of a non-human targetmonoclonal antibody fused in frame to a pool of individual humanframeworks. A human framework library that contains genes representativeof all known heavy and light chain human germline sequences can beutilized. The resulting combinatorial libraries can then be screened forbinding to antigens of interest. This approach can allow for theselection of the most favourable combinations of fully human frameworksin terms of maintaining the binding activity to the parental antibody.Humanised antibodies can then be further optimized by a variety oftechniques.

For full-length antibody molecules, the immunoglobulin genes can beobtained from genomic DNA or mRNA of hybridoma cell lines. The antibodyheavy and light chains are cloned in a mammalian vector system. Assemblyis confirmed by sequencing using methods known in the art. The antibodyconstruct can be expressed in other human or mammalian host cell lines.The construct can then be validated by transient transfection assays andWestern blot analysis of the expressed antibody of interest. Stable celllines with the highest productivity can be isolated and screened usingrapid assay methods.

Human genes that encode the constant (C) regions of the humanizedantibodies, fragments and regions can be derived from a human fetalliver library by known methods. Human C region genes can be derived fromany human cell including those that express and produce humanimmunoglobulins. The human CH region can be derived from any of theknown classes or isotypes of human heavy chains, including γ, μ, α, δ,ε, and subclasses thereof, such as G1, G2, G3 and G4. Since the heavychain isotype is responsible for the various effector functions of anantibody, the choice of CH domain will be guided by the desired effectorfunctions, such as complement fixation, or activity inantibody-dependent cellular cytotoxicity (ADCC). Preferably, the CHdomain are derived from the gamma 1 (IgG1).

The human CL region can be derived from either human L chain isotype,kappa or lambda, preferably kappa.

Genes encoding human immunoglobulin C regions are obtained from humancells by standard cloning techniques (Sambrook, et al. MolecularCloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press,Cold Spring Harbor, N.Y. (1989) and Ausubel et al., eds. CurrentProtocols in Molecular Biology (1987-1993)). Human C region genes arereadily available from known clones containing genes representing thetwo types of light chains, the five classes of heavy chains andsubclasses thereof.

Chimeric antibody fragments, such as Fab and F(ab′)₂, can be prepared bydesigning a chimeric heavy chain gene that is appropriately truncated.For example, a chimeric gene encoding a heavy chain portion of anF(ab′)₂ fragment would include DNA sequences encoding the CH1 domain andhinge region of the heavy chain, followed by a translational stop codonto yield the truncated molecule.

Methods for engineering or humanizing non-human or human antibodies canbe used and are well known in the art. Generally, a humanized orengineered antibody has one or more amino acid residues from a sourcethat is non-human, e.g., but not limited to mouse, rat, rabbit,non-human primate or other mammal. These human amino acid residues areoften referred to as “import” residues, which are typically taken froman “import” variable, constant or other domain of a known humansequence. Known human Ig sequences are disclosed, e.g.,www.ncbi.nlm.nih.gov/entrez/query.fcgi; www.atcc.org/phage/hdb.html;www.sciquest.com/; www.abcam.com/;www.antibodyresource.com/onlinecomp.html;www.public.iastate.edu/.about.pedro/research_tools.html;www.mgen.uni-heidelberg.de/SD/IT/IT.html;www.whfreeman.com/immunology/CH05/kuby05.htm;www.library.thinkquest.org/12429/Immune/Antibody.html;www.hhmi.org/grants/lectures/1996/vlab/;www.path.cam.ac.uk/.about.mrc7/mikeimages.html;www.antibodyresource.com/; mcb.harvard.edu/BioLinks/Immunology.html.www.immunologylink.com/; pathbox.wustl.edu/.about.hcenterindex.html;www.biotech.ufl.edu/.about.hcl/; www.pebio.com/pa/340913/340913.html;www.nal.usda.gov/awic/pubs/antibody/;www.m.ehime-u.ac.jp/.about.yasuhito/Elisa.html;www.biodesign.com/table.asp; www.icnet.uk/axp/facs/davies/links.html;www.biotech.ufl.edu/.about.fccl/protocol.html;www.isac-net.org/sites_geo.html;aximt1.imt.uni-marburg.de/.about.rek/AEPStart.html;baserv.uci.kun.nV.about.jraats/links1.html;www.recab.uni-hd.de/immuno.bme.nwvu.edu/;www.mrc-cpe.cam.ac.uk/imt-doc/public/INTRO.html;www.ibt.unam.mx/vir/V_mice.html; imgt.cnusc.fr.8104/;www.biochem.ucl.ac.uk/.about.martin/abs/index.html;antibody.bath.ac.uk/; abgen.cvm.tamu.edu/lab/wwwabgen.html;www.unizh.ch/.about.honegger/AHOseminar/Slide01.html;www.cryst.bbk.ac.uk/.about.ubcg07s/;www.nimr.mrc.ac.uk/CC/ccaewg/ccaewg.htm;www.path.cam.ac.uk/.about.mrc7/humanisation/TAHHP.html;www.ibt.unam.mx/vir/structure/stat_aim.html;www.biosci.missouri.edu/smithgp/index.html;www.cryst.bioc.cam.ac.uk/.about.fmolina/Web-pages/Pept/spottech.html;www.jerini.de/fr_products.htm; www.patents.ibm.con/ibm.html. Kabat etal. Sequences of Proteins of Immunological Interest, U.S. Dept. Health(1983), each entirely incorporated herein by reference.

Such imported sequences can be used to reduce immunogenicity or reduce,enhance or modify binding, affinity, on-rate, off-rate, avidity,specificity, half-life, or any other suitable characteristic, as knownin the art. Generally, part or all of the non-human or human CDRsequences are maintained while the non-human sequences of the variableand constant regions are replaced with human or other amino acids.

Antibodies can also optionally be humanized with retention of highaffinity for the antigen and other favorable biological properties. Toachieve this goal, humanized antibodies can be optionally prepared by aprocess of analysis of the parental sequences and various conceptualhumanized products using three-dimensional models of the parental andhumanized sequences. Three-dimensional immunoglobulin models arecommonly available and are familiar to those skilled in the art.Computer programs are available which illustrate and display probablethree-dimensional conformational structures of selected candidateimmunoglobulin sequences. Inspection of these displays permits analysisof the likely role of the residues in the functioning of the candidateimmunoglobulin sequence, i.e., the analysis of residues that influencethe ability of the candidate immunoglobulin to bind its antigen. In thisway, FR residues can be selected and combined from the consensus andimport sequences so that the desired antibody characteristic, such asincreased affinity for the target antigen(s), is achieved.

In general, the CDR residues are directly and most substantiallyinvolved in influencing antigen binding. Humanization or engineering theantibody can be performed using any known method, such as but notlimited to those described in Winter et al., Nature 321:522 (1986);Riechmann et al., Nature 332:323 (1988); Verhoeyen et al., Science239:1534 (1988)), Sims et al., J. Immunol. 151: 2296 (1993); Chothia andLesk, J. Mol. Biol. 196:901 (1987), Carter et al., Proc. Natl. Acad.Sci. U.S.A. 89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993),U.S. Pat. Nos. 5,723,323, 5,976,862, 5,824,514, 5,817,483, 5,814,476,5,763,192, 5,723,323, 5,766,886, 5,714,352, 6,204,023, 6,180,370,5,693,762, 5,530,101, 5,585,089, 5,225,539; 4,816,567, PCT/: US98/16280,US96/18978, US91/09630, US91/05939, US94/01234, GB89/01334, GB91/01134,GB92/01755; WO90/14443, WO90/14424, WO90/14430, EP 229246.

The human constant region of the humanized antibody can be of any classor isotype (IgG, IgA, IgM, IgE, IgD, etc.) and can comprise a kappa orlambda light chain. In one embodiment, the human constant regioncomprises an IgG heavy chain or defined fragment, for example, at leastone of the IgG subclasses, IgG1, IgG2, IgG3 or IgG4.

Labelled Antibodies Antibodies of the invention may be labelled with adetectable or functional label. Detectable labels include radiolabelssuch as [¹³¹I] or [⁹⁹Tc], which may be attached to antibodies of theinvention using conventional chemistry known in the art ofradioimmunoconjugates. Labels also include enzyme labels such ashorseradish peroxidase. Labels further include chemical moieties, suchas biotin, which may be detected via binding to a specific cognatedetectable moiety, e.g. labelled avidin or streptavidin. Preferably, thelabels include fluorescent labels such as FITC.

Organic Moiety

The modified antibodies and antigen-binding fragments can comprise oneor more organic moieties that are covalently bonded, directly orindirectly, to the antibody. Each organic moiety that is bonded to anantibody or antigen-binding fragment described herein can independentlybe a hydrophilic polymeric group, a fatty acid group or a fatty acidester group. As used herein, the term “fatty acid” encompassesmono-carboxylic acids and di-carboxylic acids. A “hydrophilic polymericgroup,” as the term is used herein, refers to an organic polymer that ismore soluble in water than in octane. For example, poly-lysine is moresoluble in water than in octane. Thus, an antibody modified by thecovalent attachment of poly-lysine is encompassed by the presentdisclosure. Hydrophilic polymers suitable for modifying antibodiesdescribed herein can be linear or branched and include, for example,poly-alkane glycols, e.g., polyethylene glycol (PEG),monomethoxy-polyethylene glycol (mPEG), PPG and the like, carbohydrates(e.g., dextran, cellulose, oligosaccharides, polysaccharides and thelike), polymers of hydrophilic amino acids (e.g., poly-lysine,poly-arginine, poly-aspartate and the like), poly-alkane oxides (e.g.,polyethylene oxide, polypropylene oxide and the like) and polyvinylpyrolidone. Preferably, the hydrophilic polymer that modifies theantibody described herein has a molecular weight of about 800 to about150,000 Daltons as a separate molecular entity. For example PEG5000 andPEG20,000, wherein the subscript is the average molecular weight of thepolymer in Daltons, can be used. The hydrophilic polymeric group can besubstituted with one to about six alkyl, fatty acid or fatty acid estergroups. Hydrophilic polymers that are substituted with a fatty acid orfatty acid ester group can be prepared by employing suitable methods.For example, a polymer comprising an amine group can be coupled to acarboxylate of the fatty acid or fatty acid ester, and an activatedcarboxylate (e.g., activated with N,N-carbonyl di-imidazole) on a fattyacid or fatty acid ester can be coupled to a hydroxyl group on apolymer.

Fatty acids and fatty acid esters suitable for modifying antibodiesdescribed herein can be saturated or can contain one or more units ofunsaturation. Fatty acids that are suitable for modifying antibodiesdescribed herein include, for example, n-dodecanoate (C12, laurate),n-tetradecanoate (C14, myristate), n-octadecanoate (C18, stearate),n-eicosanoate (C20, 5 arachidate), n-docosanoate (C22, behenate),n-triacontanoate (C30), n-tetracontanoate (C40), cis-δ 9-octadecanoate(C18, oleate), all cis-δ 5,8,11,14-eicosatetraenoate (C20,arachidonate), octanedioic acid, tetradecanedioic acid, octadecanedioicacid, docosanedioic acid, and the like. Suitable fatty acid estersinclude mono-esters of dicarboxylic acids that comprise a linear orbranched lower alkyl group. The lower alkyl group can comprise from oneto about twelve, preferably one to about six, carbon atoms.

The modified human antibodies and antigen-binding fragments can beprepared using suitable methods, such as by reaction with one or moremodifying agents. A “modifying agent” as the term is used herein, refersto a suitable organic group (e.g., hydrophilic polymer, a fatty acid, afatty acid ester) that comprises an activating group. An “activatinggroup” is a chemical moiety or functional group that can, underappropriate conditions, react with a second chemical group therebyforming a covalent bond between the modifying agent and the secondchemical group. For example, amine-reactive activating groups includeelectrophilic groups such as tosylate, mesylate, halo (chloro, bromo,fluoro, iodo), N-hydroxysuccinimidyl esters (NHS), and the like.Activating groups that can react with thiols include, for example,maleimide, iodoacetyl, acrylolyl, pyridyl disulfides,5-thiol-2-nitrobenzoic acid thiol (TNB-thiol), and the like. An aldehydefunctional group can be coupled to amine- or hydrazide-containingmolecules, and an azide group can react with a trivalent phosphorousgroup to form phosphoramidate or phosphorimide linkages. Suitablemethods to introduce activating groups into molecules are known in theart (see for example, Hemanson, G. T., Bioconjugate Techniques, AcademicPress: San Diego, Calif. (1996)). An activating group can be bondeddirectly to the organic group (e.g., hydrophilic polymer, fatty acid,fatty acid ester), or through a linker moiety, for example a divalentC1-C12 group wherein one or more carbon atoms can be replaced by aheteroatom such as oxygen, nitrogen or sulfur. Suitable linker moietiesinclude, for example, tetra-ethylene glycol, —(CH₂)₃—, —NH—(CH₂)₆—NH—,—(CH₂)₂—NH— and —CH₂—O—CH₂—CH₂—O—CH₂—CH₂—O—CH—NH—. Modifying agents thatcomprise a linker moiety can be produced, for example, by reacting amono-Boc-alkyldiamine (e.g., mono-Boc-ethylenediamine,mono-Boc-diaminohexane) with a fatty acid in the presence of1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) to form an amidebond between the free amine and the fatty acid carboxylate. The Bocprotecting group can be removed from the product by treatment withtrifluoroacetic acid (TFA) to expose a primary amine that can be coupledto another carboxylate as described, or can be reacted with maleicanhydride and the resulting product cyclized to produce an activatedmaleimido derivative of the fatty acid. (See, for example, Thompson, etal., WO 92/16221).

The modified antibodies can be produced by reacting a human antibody orantigen-binding fragment with a modifying agent. For example, theorganic moieties can be bonded to the antibody in a non-site specificmanner by employing an amine-reactive modifying agent, for example, anNHS ester of PEG. Modified human antibodies or antigen-binding fragmentscan also be prepared by reducing disulfide bonds (e.g., intra-chaindisulfide bonds) of an antibody or antigen-binding fragment. The reducedantibody or antigen-binding fragment can then be reacted with athiol-reactive modifying agent to produce the modified antibodydescribed herein. Modified human antibodies and antigen-bindingfragments comprising an organic moiety that is bonded to specific sitesof an antibody described herein can be prepared using suitable methods,such as reverse proteolysis (Fisch et al., Bioconjugate Chem., 3:147-153(1992); Werien et al., Bioconjugate Chem., 5:411-417 (1994); Kumaran etal., Protein Sci. 6(10):2233-2241 (1997); Itoh et al., Bioorg. Chem.,24(1): 59-68 (1996); Capellas et al., Biotechnol. Bioeng., 56(4):456-463(1997)), and the methods described in Hermanson, G. T., BioconjugateTechniques, Academic Press: San Diego, Calif. (1996).

Immunoconjugates

The invention also provides immunoconjugates comprising an anti-Axlantibody herein conjugated to one or more cytotoxic agents, such aschemotherapeutic agents or drugs, growth inhibitory agents, toxins(e.g., protein toxins, enzymatically active toxins of bacterial, fungal,plant, or animal origin, or fragments thereof), or radioactive isotopes.

In one embodiment, an immunoconjugate is an antibody-drug conjugate(ADC) in which an antibody is conjugated to one or more drugs, includingbut not limited to a maytansinoid (see U.S. Pat. Nos. 5,208,020,5,416,064 and European Patent EP 0 425 235 B1); an auristatin such asmonomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see U.S.Pat. Nos. 5,635,483 and 5,780,588, and 7,498,298); a dolastatin; acalicheamicin or derivative thereof (see U.S. Pat. Nos. 5,712,374,5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode etal., Cancer Res. 58:2925-2928 (1998)); an anthracycline such asdaunomycin or doxorubicin (see Kratz et al., Current Med. Chem.13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagyet al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al.,Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., J. Med.Chem. 45:4336-4343 (2002); and U.S. Pat. No. 6,630,579); methotrexate;vindesine; a taxane such as docetaxel, paclitaxel, larotaxel, tesetaxel,and ortataxel; a trichothecene; and CC1065.

In another embodiment, an immunoconjugate comprises an antibody asdescribed herein conjugated to an enzymatically active toxin or fragmentthereof, including but not limited to diphtheria toxin A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (P API, P APII, and PAP-S), Momordica Charantiainhibitor, curcin, crotin, Sapaonaria Officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

In another embodiment, an immunoconjugate comprises an antibody asdescribed herein conjugated to a radioactive atom to form aradioimmunoconjugate. A variety of radioactive isotopes are availablefor the production of radioimmunoconjugates. Examples include [²¹¹At],[¹³¹I] [¹²⁵I][⁹⁰Y], [¹⁸⁶Re], [¹⁸⁸Re], [¹⁵³Sm], [²¹²Bi], [³²P], [²¹²Pb]and radioactive isotopes of Lu. When the radioimmunoconjugate is usedfor detection, it may comprise a radioactive atom for scintigraphicstudies, for example [⁹⁹Tc] or [¹²³I], or a spin label for nuclearmagnetic resonance (NMR) imaging (also known as magnetic resonanceimaging, MRI), such as iodine-123 again, iodine-131, indium-111,fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese oriron.

Conjugates of an antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MXDTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026. Thelinker may be a “cleavable linker” facilitating release of a cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photo-labile linker, dimethyl linker ordisulfide-containing linker (Chari et al., Cancer Res. 52: 127-131(1992); U.S. Pat. No. 5,208,020) may be used.

The immunoconjugates or ADCs herein expressly contemplate, but are notlimited to such conjugates prepared with cross-linker reagentsincluding, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS,MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS,sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB(succinimidyl-(4-vinylsulfone)benzoate) which are commercially available(e.g., from Pierce Biotechnology, Inc., Rockford, Ill., U.S.A).

Glycosylation Variants

In certain embodiments, an antibody provided herein is altered toincrease or decrease the extent to which the antibody is glycosylated.Addition or deletion of glycosylation sites to an antibody may beconveniently accomplished by altering the amino acid sequence such thatone or more glycosylation sites is created or removed.

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH2 domain of the Fcregion. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). Theoligosaccharide may include various carbohydrates, e.g., mannose,N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as afucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some embodiments, modifications of theoligosaccharide in an antibody of the invention may be made in order tocreate antibody variants with certain improved properties.

In one embodiment, antibody variants are provided having a carbohydratestructure that lacks fucose attached (directly or indirectly) to an Fcregion. For example, the amount of fucose in such antibody may be from1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amountof fucose is determined by calculating the average amount of fucosewithin the sugar chain at Asn297, relative to the sum of allglycostructures attached to Asn297 (e. g., complex, hybrid and highmannose structures) as measured by MALDI-TOF mass spectrometry, asdescribed in WO 2008/077546, for example. Asn297 refers to theasparagine residue located at about position 297 in the Fc region (Eunumbering of Fc region residues); however, Asn297 may also be locatedabout ±3 amino acids upstream or downstream of position 297, i.e.,between positions 294 and 300, due to minor sequence variations inantibodies. Such fucosylation variants may have improved ADCC function.See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L.); US2004/0093621 (Kyowa Haklw Kogyo Co., Ltd). Examples of publicationsrelated to “defucosylated” or “fucose-deficient” antibody variantsinclude: US2003/01571; WO2000/61739; WO2001/29246; US2003/0115614;US2002/0164328; US2004/0093621; US2004/0132140; US2004/0110704;US2004/0110282; US2004/0109865; WO2003/085119; WO2003/084570;WO2005/035586; WO2005/035778; WO2005/053742; WO2002/031140; Okazaki etal. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech.Bioeng. 87: 614 (2004).

Examples of cell lines capable of producing defucosylated antibodiesinclude Lecl3 CHO cells deficient in protein fucosylation (Ripka et al.Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No US2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Adams et al.,especially at Example 11), and knockout cell lines, such asalpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g.,Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al.,Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003/085107).

Antibodies variants are further provided with bisected oligosaccharides,e.g., in which a biantennary oligosaccharide attached to the Fc regionof the antibody is bisected by GlcNAc. Such antibody variants may havereduced fucosylation and/or improved ADCC function. Examples of suchantibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet etal.); U.S. Pat. No. 6,602,684 (Umana et al.); and US2005/0123546 (Umanaet al.). Antibody variants with at least one galactose residue in theoligosaccharide attached to the Fe region are also provided. Suchantibody variants may have improved CDC function. Such antibody variantsare described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964(Raju, S.); and WO 1999/22764 (Raju, S.).

Fc Region Variants

In certain embodiments, one or more amino acid modifications may beintroduced into the Fc region of an antibody provided herein, therebygenerating an Fc region variant. The Fc region variant may comprise ahuman Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fcregion) comprising an amino acid modification (e.g. a substitution) atone or more amino acid positions.

In certain embodiments, the invention contemplates an antibody variantthat possesses some but not all effector functions, which make it adesirable candidate for applications in which the half-life of theantibody in vivo is important yet certain effector functions (such ascomplement fixation and ADCC) are unnecessary or deleterious. In vitroand/or in vivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks Fcγ binding (hence likely lacking ADCC activity), butretains FcRn binding ability. The primary cells for mediating ADCC, NKcells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII andFcγRIII. FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991).Non-limiting examples of in vitro assays to assess ADCC activity of amolecule of interest is described in U.S. Pat. No. 5,500,362 (see, e.g.Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) andHellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985);U.S. Pat. No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med.166:1351-1361 (1987)). Alternatively, non-radioactive assay methods maybe employed (see, for example, ACTI™ non-radioactive cytotoxicity assayfor flow cytometry (CellTechnology, Inc. Mountain View, Calif.; andCytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, Wis.).Useful effector cells for such assays include peripheral bloodmononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively,or additionally, ADCC activity of the molecule of interest may beassessed in vivo, e.g., in an animal model such as that disclosed inClynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q bindingassays may also be carried out to confirm that the antibody is unable tobind C1q and hence lacks complement-dependent cytotoxicity (CDC)activity. See, e.g., C1q and C3c binding ELISA in WO2006/029879 andWO2005/100402. To assess complement activation, a CDC assay may beperformed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods202:163 (1996); Cragg, M. S. et al., Blood 101:1045-1052 (2003); andCragg, M. S. and M. J. Glennie, Blood 103:2738-2743 (2004)). FcRnbinding and in vivo clearance/half-life Fc determinations can also beperformed using methods known in the art (see, e.g., Petkova, S. B. etal., Int'l. Immunol. 18(12): 1759-1769 (2006)).

Antibodies with reduced effector function include those withsubstitution of one or more of Fc region residues 238, 265, 269, 270,297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fcmutants with substitutions at two or more of amino acid positions 265,269, 270, 297 and 327, including the so-called “DANA” Fc mutant withsubstitution of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581).

Certain antibody variants with improved or diminished binding to FcRsare described (see, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, andShields et al., J. Biol. Chem. 9(2): 6591-6604 (2001)).

In certain embodiments, an antibody variant comprises an Fc region withone or more amino acid substitutions, that improve ADCC activity, e.g.,substitutions at positions 298, 333, and/or of the Fc region (EUnumbering of residues).

In some embodiments, alterations are made in the Fc region that resultin altered (i.e., either improved or diminished) C1q binding and/or CDCactivity, e.g., as described in U.S. Pat. No. 6,194,551, WO 99/51642,and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

Antibodies with increased half-lives and improved binding to theneonatal Fc receptor (FcRn), that is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) andKim et al., J. Immunol. 24:249 (1994)), are described inUS2005/0014934A1 (Hinton et al.). Those antibodies comprise an Fc regionwith one or more substitutions therein that improve binding of the Fcregion to FcRn. Such Fc variants include those with substitutions at oneor more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307,311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434,e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. Nos.5,648,260; 5,624,821; and WO 94/29351 concerning other examples of Fcregion variants.

Cysteine Engineered Antibody Variants

In certain embodiments, it may be desirable to create cysteineengineered antibodies, e.g., “thioMAbs,” in which one or more residuesof an antibody are substituted with cysteine residues.

In particular embodiments, the substituted residues occur at accessiblesites of the antibody. By substituting those residues with cysteine,reactive thiol groups are thereby positioned at accessible sites of theantibody and may be used to conjugate the antibody to other moieties,such as drug moieties or linker-drug moieties, to create animmunoconjugate, as described further herein. In certain embodiments,any one or more of the following residues may be substituted withcysteine: V205 (Kabat numbering) of the light chain; A 118 (EUnumbering) of the heavy chain; and S400 (EU numbering) of the heavychain Fc region. Cysteine engineered antibodies may be generated asdescribed, e.g., in U.S. Pat. No. 7,521,541.

Methods of Diagnosis and Treatment

Antibodies of the present invention are designed to be used in methodsof diagnosis or treatment in human or animal subjects, preferably human.

Accordingly, further aspects of the invention provide methods ofdiagnosis comprising administration of an antibody as provided, with oneor more reagents e.g. conjugated to a detectable label such as FITC. Theantibody as provided may be used in the development of a rapid andreliable test for cancer cells derived from biopsied tissue. Forexample, the antibody may be used as a test for metastatic cancer cells,such as circulating tumour cells, that may be found circulating in bodyfluids such as blood or lymph. Other cancers of interest include breast,lung, gastric, head and neck, colorectal, renal, pancreatic, uterine,hepatic, bladder, endometrial and prostate cancers as well as lymphomas(e.g., non-Hodgkin's lymphoma, NHL) and leukemia (particularly acutemyeloid leukemia, AML).

Further aspects of the invention provide methods of treatment comprisingadministration of an antibody as provided, pharmaceutical compositionscomprising such an antibody, the antibody as described herein for use ina method of treatment, the antibody as described herein for use in amethod of treatment of particular clinical indications described herein,and use of such an antibody in the manufacture of a medicament foradministration, for example in a method of making a medicament orpharmaceutical composition comprising formulating the antibody with apharmaceutically acceptable excipient.

Clinical Indications

Clinical indications in which an antibody with high specificity forhuman Axl may be used to provide therapeutic benefit include anycondition in which Axl is overexpressed, or wherein Axl antagonism willprovide a clinical benefit. These include immune disorders,cardiovascular disorders, thrombosis, diabetes, immune checkpointdisorders, fibrotic disorders (fibrosis), or proliferative diseases suchas cancer, particularly metastatic cancer. Furthermore, Axl is known toplay a role in many cancers of epithelial origin.

Fibrotic disorders of interest include strabmisus, scleroderma, keloid,Nephrogenic systemic fibrosis, pulmonary fibrosis, idiopathic pulmonaryfibrosis (IPF), cystic fibrosis (CF), systemic sclerosis, cardiacfibrosis, non-alcoholic steatohepatitis (NASH), other types of liverfibrosis, primary biliary cirrhosis, renal fibrosis, cancer, andatherosclerosis. In these diseases, the chronic development of fibrosisin tissue leads to marked alterations in the architecture of theaffected organs and subsequently cause defective organ function. As aresult of this process of sustained attrition to organs, many diseasesthat involve fibrosis are often progressive conditions and have a poorlong-term prognosis (see Rockey, D. C., Bell, P. D. and Hill, J. A.(2015), N. Engl. Med., Vol. 372, pp. 1138-1149).

Immune checkpoint disorders of interest include: Chronic viralinfections, Melanoma, Colorectal cancer, Breast cancer, Ovarian cancer,Non-small cell lung cancer (NSCLC), Prostate cancer, Renal cell cancer,Pancreatic cancer, Esophagus cancer, Bladder cancer, Myeloma, Kidneycancer, Bladder cancer, Brain tumor, and Lymphoma.

Cancers of interest include: leukaemias such as but not limited to,acute leukemia, acute lymphocytic leukemia, acute myelocytic leukaemiassuch as myeloblastic, promyelocytic, myelomonocytic, monocytic,erythroleukaemia leukaemias and myelodysplastic syndrome, chronicleukaemias such as but not limited to, chronic myelocytic (granulocytic)leukemia, chronic lymphocytic leukemia, hairy cell leukemia;polycythemia vera; lymphomas such as but not limited to Hodgkin'sdisease, non-Hodgkin's disease; multiple myelomas such as but notlimited to smoldering multiple myeloma, nonsecretory myeloma,osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma andextramedullary plasmacytoma; Waldenstrom's macroglobulinemia; monoclonalgammopathy of undetermined significance; benign monoclonal gammopathy;heavy chain disease; bone and connective tissue sarcomas such as but notlimited to bone sarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma,malignant giant cell tumor, fibrosarcoma of bone, chordoma, periostealsarcoma, soft-tissue sarcomas, angiosarcoma (hemangiosarcoma),fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma,lymphangiosarcoma, metastatic cancers, neurilemmoma, rhabdomyosarcoma,synovial sarcoma; brain tumors such as but not limited to, glioma,glioblastoma, astrocytoma, brain stem glioma, ependymoma,oligodendroglioma, nonglial tumor, acoustic neurinoma,craniopharyngioma, medulloblastoma, meningioma, pineocytoma,pineoblastoma, primary brain lymphoma; breast cancer, including, but notlimited to, adenocarcinoma, lobular (small cell) carcinoma, intraductalcarcinoma, medullary breast cancer, mucinous breast cancer, tubularbreast cancer, papillary breast cancer, primary cancers, Paget'sdisease, and inflammatory breast cancer; adrenal cancer such as but notlimited to pheochromocytoma, and adrenocortical carcinoma; thyroidcancer such as but not limited to papillary or follicular thyroidcancer, Medullary thyroid carcinoma, medullary thyroid cancer andanaplastic thyroid cancer; GIST-gastrointestinal stromal tumor;pancreatic cancer such as but not limited to, insulinoma, gastrinoma,glucagonoma, vipoma, somatostatin-secreting tumor, and carcinoid orislet cell tumor; pituitary cancers such as but limited to Cushing'sdisease, prolactin-secreting tumor, acromegaly, and diabetes insipius;eye cancers such as but not limited to ocular melanoma such as irismelanoma, choroidal melanoma, and cilliary body melanoma, andretinoblastoma; vaginal cancers such as squamous cell carcinoma,adenocarcinoma, and melanoma; vulvar cancer such as squamous cellcarcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, andPaget's disease; cervical cancers such as but not limited to, squamouscell carcinoma, and adenocarcinoma; uterine cancers such as but notlimited to endometrial carcinoma and uterine sarcoma; ovarian cancerssuch as but not limited to, ovarian epithelial carcinoma, borderlinetumor, germ cell tumor, and stromal tumor; esophageal cancers such asbut not limited to, squamous cancer, adenocarcinoma, adenoid cycticcarcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma,melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small cell)carcinoma; stomach cancers such as but not limited to, adenocarcinoma,fungating (polypoid), ulcerating, superficial spreading, diffuselyspreading, malignant lymphoma, liposarcoma, fibrosarcoma, andcarcinosarcoma; colon cancers; rectal cancers; liver cancers such as butnot limited to hepatocellular carcinoma and hepatoblastoma, gallbladdercancers such as adenocarcinoma; cholangiocarcinomas such as but notlimited to pappillary, nodular, and diffuse; lung cancers such asnon-small cell lung cancer (NSCLC), squamous cell carcinoma (epidermoidcarcinoma), adenocarcinoma, large-cell carcinoma and small-cell lungcancer (SCLC); testicular cancers such as but not limited to germinaltumor, seminoma, anaplastic, classic (typical), spermatocytic,nonseminoma, embryonal carcinoma, teratoma carcinoma, choriocarcinoma(yolk-sac tumor), prostate cancers such as but not limited to,adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; genital cancerssuch as penile cancer; oral cancers such as but not limited to squamouscell carcinoma; basal cancers; salivary gland cancers such as but notlimited to adenocarcinoma, mucoepidermoid carcinoma, and adenoidcysticcarcinoma; pharynx cancers such as but not limited to squamous cellcancer, and verrucous; skin cancers such as but not limited to, basalcell carcinoma, squamous cell carcinoma and melanoma, superficialspreading melanoma, nodular melanoma, lentigo malignant melanoma, acrallentiginous melanoma; kidney cancers such as but not limited to renalcell cancer, Clear cell renal cell carcinoma, adenocarcinoma,hypemephroma, fibrosarcoma, transitional cell cancer (renal pelvisand/or ureter); Wilms' tumor; bladder cancers such as but not limited totransitional cell carcinoma, squamous cell cancer, adenocarcinoma,carcinosarcoma. In addition, cancers include myxosarcoma, osteogenicsarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma,synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma,bronchogenic carcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma and papillary adenocarcinomas.Preferably, the cancer is selected from breast, melanoma, prostate,ovarian, colorectal, lung or glioma cancer. More preferably, the canceris metastatic breast or lung cancer. The targeting and treatment ofcirculating tumour cells is envisaged.

The treatment of metastatic cancer depends on where the primary tumouris located. When breast cancer spreads to the lungs, for example, itremains a breast cancer and the treatment is determined by themetastatic cancer origin within the breast, not by the fact that it isnow in the lung. About 5 percent of the time, metastatic cancer isdiscovered but the primary tumour cannot be identified. The treatment ofthese metastatic cancers is dictated by their location rather than theirorigin. Metastatic cancers are named by the tissue of the originaltumour (if known). For example, a breast cancer that has spread to thebrain is called metastatic breast cancer to the brain.

Anti-Axl treatment in accordance with the present invention may be usedto provide clear benefit for patients with conditions where Axl isoverexpressed, or wherein Axl antagonism will provide a clinicalbenefit. Treatment may be given by injection (e.g. intravenously) or bylocal delivery methods. The antibody as provided may be used to directthe delivery of pharmaceutical compositions to the target tissue, orsystemically in order to target, for example, Circulating Tumour Cells(CTCs) or other metastatic cells.

In a further aspect of the invention, there is provided a method ofinhibiting Cancer Stem Cells in a subject, the method comprising ofcontacting the subject with an antibody (or conjugate thereof) asdescribed herein. Antibodies and conjugates for use in such a method arealso envisaged.

EGFR Antagonism

The invention also provides methods of inhibiting constitutive Axlactivation comprising administering to the individual an effectiveamount of any of the anti-Axl antibodies disclosed herein to inhibitconstitutive Axl.

In one aspect, the invention provides methods for treating a subjectsuffering from a cancer associated with an EGFR activating mutation oran EGFR gene amplification, wherein the subject has developed aresistance to treatment with an EGFR antagonist, comprising determiningwhether the subject has Axl expression, an Axl activating mutation or anAxl gene amplification, and administering to those subjects having anAxl activating mutation or an Axl gene amplification an EGFR antagonistand any of the anti-Axl antibodies described herein.

In one aspect, the invention provides methods for treating a subjectsuffering from a cancer associated with an EGFR activating mutation oran EGFR gene amplification, comprising: (i) monitoring a subject beingtreated with an EGFR antagonist to determine if the subject develops Axlexpression, an Axl activating mutation or an Axl gene amplification, and(ii) modifying the treatment regimen of the subject to include any ofthe anti-Axl antibodies described herein in addition to the EGFRantagonist where the subject has developed an Axl activating mutation oran Axl gene amplification.

In one aspect, the invention provides methods for treating a subjectsuffering from a cancer associated with an EGFR activating mutation oran EGFR gene amplification, comprising: (i) monitoring a subject beingtreated with EGFR antagonist to determine if the subject develops aresistance to the inhibitor, (ii) testing the subject to determinewhether the subject has Axl expression, an Axl activating mutation or anAxl gene amplification, and (iii) modifying the treatment regimen of thesubject to include any of the anti-Axl antibodies described herein inaddition to the EGFR antagonist where the subject has an Axl activatingmutation or an Axl gene amplification.

In one aspect, the invention provides methods for evaluating an EGFRantagonist, comprising: (i) monitoring a population of subjects beingtreated with an EGFR antagonist to identify those subjects that developa resistance to the therapeutic, (ii) testing the resistant subjects todetermine whether the subjects have Axl expression, an Axl activatingmutation or an Axl gene amplification, and (iii) modifying the treatmentregimen of the subjects to include any of the anti-Axl antibodiesdescribed herein in addition to the EGFR antagonist where the subjectshave Axl expression, an Axl activating mutation or an Axl geneamplification.

In one aspect, the invention provides methods for reducing EGFRphosphorylation in a cancer cell, wherein said cancer cell has acquiredresistance to an EGFR antagonist, and wherein said cell comprises an Axlactivating mutation or an Axl gene amplification, comprising the step ofcontacting the cell with any of the anti-Axl antibodies described hereinand an EGFR antagonist.

In one aspect, the invention provides methods for reducing PBK mediatedsignaling in a cancer cell, wherein said cancer cell has acquiredresistance to an EGFR antagonist, and wherein said cell comprises Axlexpression, an Axl activating mutation or an Axl gene amplification,comprising the step of contacting the cell with any of the anti-Axlantibodies described herein and an EGFR antagonist.

In one aspect, the invention provides methods for reducing EGFR-mediatedsignaling in a cancer cell, wherein said cancer cell has acquiredresistance to an EGFR antagonist, and wherein said cell comprises Axlexpression, an Axl activating mutation or an Axl gene amplification,comprising contacting the cell with any of the anti-Axl antibodiesdescribed herein and an EGFR antagonist.

In one aspect, the invention provides methods for restoring sensitivityof a cancer cell to an EGFR antagonist, wherein said cancer cell hasacquired resistance to an EGFR antagonist, and wherein said cellcomprises Axl expression, an Axl activating mutation or an Axl geneamplification, comprising contacting the cell with any of the anti-Axlantibodies described herein and an EGFR antagonist.

In one aspect, the invention provides methods for reducing growth orproliferation of a cancer cell, wherein said cancer cell has acquiredresistance to an EGFR antagonist, and wherein said cell comprises Axlexpression, an Axl activating mutation or an Axl gene amplification,comprising the step of contacting the cell with any of the anti-Axlantibodies described herein and an EGFR antagonist.

In one aspect, the invention provides methods for increasing apoptosisof a cancer cell, wherein said cancer cell has acquired resistance to anEGFR antagonist, and wherein said cell comprises Axl expression, an Axlactivating mutation or an Axl gene amplification, comprising the step ofcontacting the cell with any of the anti-Axl antibodies described hereinand an EGFR antagonist.

In one aspect, the invention provides methods for reducing resistance ofa cancer cell to an EGFR antagonist, wherein said cancer cell hasacquired resistance to an EGFR antagonist, and wherein said cellcomprises an Axl activating mutation or an Axl gene amplification,comprising the step of contacting the cell with any of the anti-Axlantibodies described herein and an EGFR antagonist.

In one aspect, the invention provides methods for treating acquired EGFRantagonist resistance in a cancer cell, wherein said cell comprises anAxl activating mutation or an Axl gene amplification, comprisingcontacting the cell with any of the anti-Axl antibodies described hereinand an EGFR antagonist.

In some embodiments, the cancer cell is any EGFR-driven cancer. In someembodiments, the cancer cell comprises an EGFR activating mutation. Insome embodiments, the cancer cell comprises an EGFR gene amplification.In some embodiments, the EGFR gene amplification is at least 2-fold. Insome embodiments, the Axl amplification is at least 2-fold. In someembodiments, the cancer cell comprises an EGFR gene mutation associatedwith increased resistance to an EGFR antagonist. In some embodiments,the EGFR gene mutation associated with increased resistance to an EGFRantagonist is a T790M mutation of EGFR.

In some embodiments, the EGFR antagonist is a small moleculetherapeutic, a nucleic acid therapeutic, or a protein therapeutic. Insome embodiments, the EGFR antagonist is an antibody, an antisensemolecule, or a small molecule kinase inhibitor. In some embodiments, theEGFR antagonist is an EGFR kinase inhibitor selected from the groupconsisting of: gefitinib, erlotinib, cetuximab, panitumumab. In someembodiments, the EGFR antagonist is an anti-EGFR antibody selected fromthe group consisting of: cetuximab, panitumumab. In some embodiments,the nucleic acid therapeutic is a siRNA molecule.

In one aspect, the invention provides methods for identifying a subjectas a candidate for treatment with an EGFR antagonist and any of theanti-Axl antibodies described herein, wherein said subject has beentreated with an EGFR antagonist and suffers from cancer that hasacquired resistance to said EGFR antagonist, comprising detecting Axlexpression, an Axl activating mutation or Axl gene amplification in acancer cell from said subject.

In one aspect, the invention provides methods for identifying a subjectwho is being treated with an EGFR antagonist and who is at risk foracquiring resistance to said EGFR antagonist, comprising detecting thepresence of Axl expression, an Axl activating mutation or an Axl geneamplification in a cancer cell from said subject, wherein the presenceof said Axl expression, Axl activating mutation or Axl geneamplification indicates a risk for acquiring said resistance.

In one aspect, the invention provides methods for treating a subjectsuffering from a cancer that is resistant to treatment with an EGFRantagonist, comprising administering to the subject an EGFR antagonistand any of the anti-Axl antibodies described herein.

In one aspect, the invention provides methods for treating a subjectsuffering from a cancer associated with an EGFR activating mutation oran EGFR gene amplification, wherein the subject has developed aresistance to treatment with an EGFR antagonist, comprising determiningwhether the subject has Axl expression, such as elevated Axl levelsand/or activity, and administering to those subjects having Axlexpression, such as elevated Axl activity an EGFR antagonist and any ofthe anti-Axl antibodies described herein.

In one aspect, the invention provides methods for treating a subjectsuffering from a cancer associated with an EGFR activating mutation oran EGFR gene amplification, comprising: (i) monitoring a subject beingtreated with an EGFR antagonist to determine if the subject develops Axlexpression, such as elevated levels and/or Axl activity, and (ii)modifying the treatment regimen of the subject to include any of theanti-Axl antibodies described herein in addition to the EGFR antagonistwhere the subject has developed Axl expression, such as elevated Axllevels and/or activity.

In one aspect, the invention provides methods for treating a subjectsuffering from a cancer associated with an EGFR activating mutation oran EGFR gene amplification, comprising: (i) monitoring a subject beingtreated with EGFR antagonist to determine if the subject develops aresistance to the inhibitor, (ii) testing the subject to determinewhether the subject has Axl expression, such as elevated Axl levelsand/or activity, and (iii) modifying the treatment regimen of thesubject to include any of the anti-Axl antibodies described herein inaddition to the EGFR antagonist where the subject has elevated Axllevels and/or activity.

In another aspect, the invention provides a method for (i) restoring thesensitivity of a cancer cell to an EGFR antagonist, (ii) reducingresistance of a cancer cell to an EGFR antagonist, and/or (iii) treatingacquired EGFR antagonist resistance in a cancer cell, by contacting thecell with an EGFR antagonist and any of the anti-Axl antibodiesdescribed herein.

In exemplary embodiments, the cancer cell has acquired a resistance toan EGFR antagonist and comprises elevated levels of Axl activity and/orexpression, e.g., associated with an activating mutation in the Axlgene, an Axl gene amplification, or Gas6 mediated Axl activation. Themethods disclosed herein may be used to restore the sensitivity, reducethe resistance, and/or treat an acquired resistance, of a cancer cell.

In another aspect, the invention provides a method for reducing growthand/or proliferation of a cancer cell, or increasing apoptosis of acancer cell, by contacting the cell with an EGFR antagonist and any ofthe anti-Axl antibodies described herein. In exemplary embodiments, thecancer cell has acquired a resistance to an EGFR antagonist andcomprises elevated Axl activity and/or expression, e.g., associated withan activating mutation in the Axl gene, an Axl gene amplification, orGas6 mediated Axl activation.

Pharmaceutical Compositions

Antibodies of the present invention will usually be administered in theform of a pharmaceutical composition, that may comprise at least onecomponent in addition to the antibody.

Thus pharmaceutical compositions according to the present invention, andfor use in accordance with the present invention, may comprise, inaddition to active ingredient, a pharmaceutically acceptable excipient,carrier, buffer, stabiliser or other materials well known to thoseskilled in the art. Such materials should be non-toxic and should notinterfere with the efficacy of the active ingredient. The precise natureof the carrier or other material will depend on the route ofadministration, which may be oral, or by injection, e.g. intravenous.The pharmaceutical compositions may be for human or animal usage inhuman and veterinary medicine.

Examples of such suitable excipients for the various different forms ofpharmaceutical compositions described herein may be found in the“Handbook of Pharmaceutical Excipients”, 2nd Edition, (1994), Edited byA Wade and P J Weller.

Acceptable carriers or diluents for therapeutic use are well known inthe pharmaceutical art, and are described, for example, in Remington'sPharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).Examples of suitable carriers include lactose, starch, glucose,methylcellulose, magnesium stearate, mannitol, sorbitol and the like.Examples of suitable diluents include ethanol, glycerol, water andbuffered saline.

The choice of pharmaceutical carrier, excipient or diluent can beselected with regard to the intended route of administration andstandard pharmaceutical practice. The pharmaceutical compositions maycomprise as, or in addition to, the carrier, excipient or diluent anysuitable binder(s), lubricant(s), suspending agent(s), coating agent(s),solubilising agent(s), buffer(s), flavouring agent(s), surface activeagent(s), thickener(s), preservative(s) (including antioxidants) and thelike, and substances included for the purpose of rendering theformulation isotonic with the blood of the intended recipient.

Examples of suitable binders include starch, gelatine, natural sugarssuch as glucose, anhydrous lactose, free-flow lactose, beta-lactose,corn sweeteners, natural and synthetic gums, such as acacia, tragacanthor sodium alginate, carboxymethyl cellulose and polyethylene glycol.

Examples of suitable lubricants include sodium oleate, sodium stearate,magnesium stearate, sodium benzoate, sodium acetate, sodium chloride andthe like. Preservatives, stabilizers, dyes and even flavouring agentsmay be provided in the pharmaceutical composition. Examples ofpreservatives include sodium benzoate, sorbic acid and esters of phydroxybenzoic acid. Antioxidants and suspending agents may be alsoused.

Pharmaceutical formulations include those suitable for oral, topical(including dermal, buccal and sublingual), rectal or parenteral(including subcutaneous, intradermal, intramuscular and intravenous),nasal and pulmonary administration, e.g., by inhalation. The formulationmay, where appropriate, be conveniently presented in discrete dosageunits and may be prepared by any of the methods well known in the art ofpharmacy. All methods include the step of bringing into association anactive compound with liquid carriers or finely divided solid carriers orboth and then, if necessary, shaping the product into the desiredformulation. Pharmaceutical formulations suitable for oraladministration wherein the carrier is a solid are most preferablypresented as unit dose formulations such as boluses, capsules or tabletseach containing a predetermined amount of active agent. A tablet may bemade by compression or moulding, optionally with one or more accessoryingredients. Compressed tablets may be prepared by compressing in asuitable machine an active agent in a free-flowing form such as a powderor granules optionally mixed with a binder, lubricant, inert diluent,lubricating agent, surface-active agent or dispersing agent. Mouldedtablets may be made by moulding an active agent with an inert liquiddiluent. Tablets may be optionally coated and, if uncoated, mayoptionally be scored. Capsules may be prepared by filling an activeagent, either alone or in admixture with one or more accessoryingredients, into the capsule shells and then sealing them in the usualmanner. Cachets are analogous to capsules wherein an active agenttogether with any accessory ingredient(s) is sealed in a rice paperenvelope. An active agent may also be formulated as dispersiblegranules, which may for example be suspended in water beforeadministration, or sprinkled on food. The granules may be packaged,e.g., in a sachet. Formulations suitable for oral administration whereinthe carrier is a liquid may be presented as a solution or a suspensionin an aqueous or non-aqueous liquid, or as an oil-in-water liquidemulsion.

Formulations for oral administration include controlled release dosageforms, e.g., tablets wherein an active agent is formulated in anappropriate release—controlling matrix, or is coated with a suitablerelease—controlling film. Such formulations may be particularlyconvenient for prophylactic use.

Pharmaceutical formulations suitable for rectal administration whereinthe carrier is a solid are most preferably presented as unit dosesuppositories. Suitable carriers include cocoa butter and othermaterials commonly used in the art. The suppositories may beconveniently formed by admixture of an active agent with the softened ormelted carrier(s) followed by chilling and shaping in moulds.

Pharmaceutical formulations suitable for parenteral administrationinclude sterile solutions or suspensions of an active agent in aqueousor oleaginous vehicles.

Injectable preparations may be adapted for bolus injection or continuousinfusion. Such preparations are conveniently presented in unit dose ormulti-dose containers that are sealed after introduction of theformulation until required for use. Alternatively, an active agent maybe in powder form which is constituted with a suitable vehicle, such assterile, pyrogen-free water, before use.

An active compound may also be formulated as long-acting depotpreparations, which may be administered by intramuscular injection or byimplantation, e.g., subcutaneously or intramuscularly. Depotpreparations may include, for example, suitable polymeric or hydrophobicmaterials, or ion-exchange resins. Such long-acting formulations areparticularly convenient for prophylactic use.

Formulations suitable for pulmonary administration via the buccal cavityare presented such that particles containing an active compound anddesirably having a diameter in the range of 0.5 to 7 microns aredelivered in the bronchial tree of the recipient. As one possibilitysuch formulations are in the form of finely comminuted powders which mayconveniently be presented either in a pierceable capsule, suitably of,for example, gelatin, for use in an inhalation device, or alternativelyas a self-propelling formulation comprising an active agent, a suitableliquid or gaseous propellant and optionally other ingredients such as asurfactant and/or a solid diluent. Suitable liquid propellants includepropane and the chlorofluorocarbons, and suitable gaseous propellantsinclude carbon dioxide. Self-propelling formulations may also beemployed wherein an active agent is dispensed in the form of droplets ofsolution or suspension.

Such self-propelling formulations are analogous to those known in theart and may be prepared by established procedures. Suitably they arepresented in a container provided with either a manually-operable orautomatically functioning valve having the desired spraycharacteristics; advantageously the valve is of a metered typedelivering a fixed volume, for example, 25 to 100 microliters, upon eachoperation thereof.

As a further possibility, an active agent may be in the form of asolution or suspension for use in an atomizer or nebuliser whereby anaccelerated airstream or ultrasonic agitation is employed to produce afine droplet mist for inhalation.

Formulations suitable for nasal administration include preparationsgenerally similar to those described above for pulmonary administration.When dispensed such formulations should desirably have a particlediameter in the range 10 to 200 microns to enable retention in the nasalcavity; this may be achieved by, as appropriate, use of a powder of asuitable particle size or choice of an appropriate valve. Other suitableformulations include coarse powders having a particle diameter in therange 20 to 500 microns, for administration by rapid inhalation throughthe nasal passage from a container held close up to the nose, and nasaldrops comprising 0.2 to 5% w/v of an active agent in aqueous or oilysolution or suspension.

Pharmaceutically acceptable carriers are well known to those skilled inthe art and include, but are not limited to, 0.1 M and preferably 0.05 Mphosphate buffer or 0.8% saline. Additionally, such pharmaceuticallyacceptable carriers may be aqueous or non-aqueous solutions,suspensions, and emulsions. Examples of non-aqueous solvents arepropylene glycol, polyethylene glycol, vegetable oils such as olive oil,and injectable organic esters such as ethyl oleate. Aqueous carriersinclude water, alcoholic/aqueous solutions, emulsions or suspensions,including saline and buffered media. Parenteral vehicles include sodiumchloride solution, Ringer's dextrose, dextrose and sodium chloride,lactated Ringer's or fixed oils. Preservatives and other additives mayalso be present, such as, for example, antimicrobials, antioxidants,chelating agents, inert gases and the like.

Formulations suitable for topical formulation may be provided forexample as gels, creams or ointments. Such preparations may be appliede.g. to a wound or ulcer either directly spread upon the surface of thewound or ulcer or carried on a suitable support such as a bandage,gauze, mesh or the like which may be applied to and over the area to betreated.

Liquid or powder formulations may also be provided which can be sprayedor sprinkled directly onto the site to be treated, e.g. a wound orulcer. Alternatively, a carrier such as a bandage, gauze, mesh or thelike can be sprayed or sprinkle with the formulation and then applied tothe site to be treated.

According to a further aspect of the invention, there is provided aprocess for the preparation of a pharmaceutical or veterinarycomposition as described above, the process comprising bringing theactive compound(s) into association with the carrier, for example byadmixture.

In general, the formulations are prepared by uniformly and intimatelybringing into association the active agent with liquid carriers orfinely divided solid carriers or both, and then if necessary shaping theproduct. The invention extends to methods for preparing a pharmaceuticalcomposition comprising bringing an agent into association with apharmaceutically or veterinary acceptable carrier or vehicle.

Administration

The pharmaceutical compositions of the present invention may be adaptedfor oral, rectal, nasal, intrabronchial, topical (including buccal andsublingual), vaginal or parenteral (including subcutaneous,intramuscular, intravenous, intra-arterial and intradermal),intraperitoneal or intrathecal administration. Preferably, theformulation is an intravenously or subcutaneously administeredformulation.

The formulations may conveniently be presented in unit dosage form,i.e., in the form of discrete portions containing a unit dose, or amultiple or sub-unit of a unit dose. By way of example, the formulationsmay be in the form of tablets and sustained release capsules, and may beprepared by any method well known in the art of pharmacy.

Formulations for oral administration in the present invention may bepresented as: discrete units such as capsules, gellules, drops, cachets,pills or tablets each containing a predetermined amount of the activeagent; as a powder or granules; as a solution, emulsion or a suspensionof the active agent in an aqueous liquid or a non-aqueous liquid; or asan oil-in-water liquid emulsion or a water-in-oil liquid emulsion; or asa bolus etc. Preferably, these compositions contain from 1 to 250 mg andmore preferably from 10-100 mg, of active ingredient per dose.

For compositions for oral administration (e.g. tablets and capsules),the term “acceptable carrier” includes vehicles such as commonexcipients e.g. binding agents, for example syrup, acacia, gelatine,sorbitol, tragacanth, polyvinylpyrrolidone (Povidone), methylcellulose,ethylcellulose, sodium carboxymethylcellulose,hydroxypropyl-methylcellulose, sucrose and starch; fillers and carriers,for example corn starch, gelatine, lactose, sucrose, microcrystallinecellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride andalginic acid; and lubricants such as magnesium stearate, sodium stearateand other metallic stearates, glycerol stearate stearic acid, siliconefluid, talc waxes, oils and colloidal silica. Flavouring agents such aspeppermint, oil of wintergreen, cherry flavouring and the like can alsobe used. It may be desirable to add a colouring agent to make the dosageform readily identifiable. Tablets may also be coated by methods wellknown in the art.

A tablet may be made by compression or moulding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active agent in a free flowingform such as a powder or granules, optionally mixed with a binder,lubricant, inert diluent, preservative, surface-active or dispersingagent. Moulded tablets may be made by moulding in a suitable machine amixture of the powdered compound moistened with an inert liquid diluent.The tablets may be optionally be coated or scored and may be formulatedso as to provide slow or controlled release of the active agent.

Other formulations suitable for oral administration include lozengescomprising the active agent in a flavoured base, usually sucrose andacacia or tragacanth; pastilles comprising the active agent in an inertbase such as gelatine and glycerine, or sucrose and acacia; andmouthwashes comprising the active agent in a suitable liquid carrier.

Other forms of administration comprise solutions or emulsions which maybe injected intravenously, intra-arterially, intrathecally,subcutaneously, intradermally, intraperitoneally or intramuscularly, andwhich are prepared from sterile or sterilisable solutions. Injectableforms typically contain between 10-1000 mg, preferably between 10-250mg, of active ingredient per dose.

The pharmaceutical compositions of the present invention may also be inform of suppositories, pessaries, suspensions, emulsions, lotions,ointments, creams, gels, sprays, solutions or dusting powders.

An alternative means of transdermal administration is by use of a skinpatch. For example, the active ingredient can be incorporated into acream consisting of an aqueous emulsion of polyethylene glycols orliquid paraffin. The active ingredient can also be incorporated, at aconcentration of between 1 and 10% by weight, into an ointmentconsisting of a white wax or white soft paraffin base together with suchstabilisers and preservatives as may be required.

Alternative formulation strategies may provide preparations suitable fororal or suppository route. The route of administration may be determinedby the physicochemical characteristics of the treatment, by specialconsiderations for the disease, to optimise efficacy or to minimiseside-effects.

A further mode of administration employs pre-coating of, or otherwiseincorporation into, indwelling devices, for which the optimal amount ofantibody will be determined by means of appropriate experiments.

An antibody molecule in some preferred embodiments of the invention is amonomeric fragment, such as Fab or scFv. Such antibody fragments mayhave the feature of a relatively short half-life.

Dosage

A person of ordinary skill in the art can easily determine anappropriate dose of one of the instant compositions to administer to asubject without undue experimentation. Typically, a physician willdetermine the actual dosage which will be most suitable for anindividual patient and it will depend on a variety of factors includingthe activity of the specific agent employed, the metabolic stability andlength of action of that agent, the age, body weight, general health,sex, diet, mode and time of administration, rate of excretion, drugcombination, the severity of the particular condition, and theindividual undergoing therapy.

In accordance with the present invention, compositions provided may beadministered to individual patients. Administration is preferably in a“therapeutically effective amount”, this being sufficient to showbenefit to a patient. Such benefit may be at least amelioration of atleast one symptom. The actual amount administered, and the rate andtime-course of administration, will depend on the nature and severity ofwhat is being treated. Prescription of treatment, e.g., decisions ondosage etc., is within the responsibility of general practitioners andother medical doctors. Appropriate doses of antibody are well known inthe art; see Ledermann J. A. et al. (1991) Int. J. Cancer 47: 659-664;Bagshawe, K. D. et al. (1991) Antibody, Immunoconjugates andRadiopharmaceuticals 4: 915-922.

The precise dose will depend upon a number of factors, including whetherthe antibody is for diagnosis or for treatment, the size and location ofthe area to be treated, the precise nature of the antibody (e.g. wholeantibody, antibody fragment or diabody), and the nature of anydetectable label or other molecule attached to the antibody. A typicalantibody dose may be administered as a bolus intravenously. Other modesof administration include intravenous infusion over several hours, toachieve a similar total cumulative dose. This is a dose for a singletreatment of an adult patient, which may be proportionally adjusted forchildren and infants, and also adjusted for other antibody formats inproportion to molecular weight. Treatments may be repeated at daily,twice-weekly, weekly or monthly intervals, at the discretion of thephysician.

The dosages disclosed herein are exemplary of the average case. Therecan of course be individual instances where higher or lower dosageranges are merited, and such are within the scope of this invention.

In accordance with this invention, an effective amount of agent may beadministered to inhibit Axl. Of course, this dosage amount will furtherbe modified according to the type of administration of the agent. Forexample, to achieve an “effective amount” for acute therapy, parenteraladministration is preferred. An intravenous infusion of the compound in5% dextrose in water or normal saline, or a similar formulation withsuitable excipients, is most effective, although an intramuscular bolusinjection is also useful. Typically, the parenteral dose will be about0.01 to about 100 mg/kg; preferably between 0.1 and 20 mg/kg, in amanner to maintain the concentration of drug in the plasma at aconcentration effective to inhibit a kinase or saturate the targetreceptor. The agents may be administered one to four times daily at alevel to achieve a total daily dose of about 0.4 to about 400 mg/kg/day.The precise amount of an active agent that is therapeutically effective,and the route by which such agent is best administered, is readilydetermined by one of ordinary skill in the art by comparing the bloodlevel of the agent to the concentration required to have a therapeuticeffect.

The agents of this invention may also be administered orally to thepatient, in a manner such that the concentration of drug is sufficientto achieve one or more of the therapeutic indications disclosed herein.Typically, a pharmaceutical composition containing the agent isadministered at an oral dose of between about 0.1 to about 50 mg/kg in amanner consistent with the condition of the patient. Preferably, theoral dose would be about 0.5 to about 20 mg/kg.

The agents of this invention may be tested in one of several biologicalassays to determine the concentration of an agent that is required tohave a given pharmacological effect.

Combination Therapy

The anti-Axl antibodies of the invention may be administered alone or incombination with other treatments, either simultaneously or sequentiallydependent upon the condition to be treated. For example, the antibodiesof the invention or conjugates thereof may be used as an anti-cancermonotherapy or in combination therapy with other cancer treatments asmentioned below. Other treatments may include the administration ofsuitable doses of pain relief drugs such as non-steroidalanti-inflammatory drugs (e.g. aspirin, ibuprofen or ketoprofen) oropiates such as morphine, or anti-emetics.

Suitable Agents for Use in Combination Therapy

These include alkylating agents, e.g., alkyl sulfonates such asbusulfan;

nitrogen mustards such as chlorambucil, cyclophosphamide, estramustine,ifosfamide, mechlorethamine, melphalan, and uramustine, ethyleneiminederivatives such as thiotepa; nitrosoureas such as carmustine,lomustine, and streptozocin, triazenes such as dacarbazine,procarbazine, and temozolamide;

platinum compounds such as cisplatin, carboplatin, oxaliplatin,satraplatin, and picoplatin onnaplatin, tetraplatin, sprioplatin,iproplatin, chloro(diethylenediamino)-platinum (II) chloride,dichloro(ethylenediamino)-platinum (II),diamino(2-ethylmalonato)platinum (II),(1,2-diaminocyclohexane)malonatoplatinum (II),(4-carboxyphthalo)-(1,2-diaminocyclohexane)platinum (II),(1,2-diaminocyclohexane)-(isocitrato)platinum (II), and(1,2-diaminocyclohexane)-cis-(pyruvato)platinum (II);

anti-metabolites, including antifolates such as methotrexate,permetrexed, raltitrexed, and trimetrexate;

pyrimidine analogs such as azacitidine, capecitabine, cytarabine,edatrexate, floxuridine, fluorouracil, gemcitabine, and troxacitabine;

purine analogs such as cladribine, chlorodeoxyadenosine, clofarabine,fludarabine, mercaptopurine, pentostatin, and thioguanine;

natural products, including antitumor antibiotics such as bleomycin,dactinomycin, mithramycin, mitomycin, mitoxantrone, porfiromycin, andanthracyclines such as daunorubicin, doxorubicin, epirubicin,idarubicin, and valrubicin;

mitotic inhibitors such as the vinca alkaloids vinblastine, vinvesir,vincristine, vindesine, and vinorelbine;

enzymes such as L-asparaginase and PEG-L-asparaginase;

microtubule polymer stabilizers such as the taxanes paclitaxel anddocetaxel;

topoisomerase I inhibitors such as the camptothecins irinotecan andtopotecan; topoisomerase II inhibitors such as podophyllotoxin,amsacrine, etoposide, teniposide, losoxantrone and actinomycin;

hormones and hormone antagonists, including androgens such asfluoxymesterone and testolactone,

anti-androgens such as bicalutamide, cyproterone, flutamide, andnilutamide;

corticosteroids such as dexamethasone and prednisone;

aromatase inhibitors such as aminoglutethimide, anastrozole, exemestane,formestane, and letrozole;

estrogens such as diethylstilbestrol;

anti-estrogens such as fulvestrant, raloxifene, tamoxifen, andtoremifine;

luteinising hormone-releasing hormone (LHRH) agonists and antagonistssuch as abarelix, buserelin, goserelin, leuprolide, histrelin,desorelin, nafarelin acetate and triptorelin;

progestins such as medroxyprogesterone acetate and megestrol acetate,and

thyroid hormones such as levothyroxine and liothyronine;

PKB pathway inhibitors, including perifosine, enzastaurin hydrochloride,and triciribine;

PI3K inhibitors such as semaphore and SF1126;

mTOR inhibitors such as rapamycin and analogues;

CDK inhibitors, including seliciclib, alvocidib, and7-hydroxystaurosponne;

COX-2 inhibitors, including celecoxib;

HDAC inhibitors, including trichostatin A, suberoylanilide hydroxamicacid, and chlamydocin;

DNA methylase inhibitors, including temozolomide; and

miscellaneous agents, including altretamine, arsenic trioxide,thalidomide, lenalidomide, gallium nitrate, levamisole, mitotane,hydroxyurea, octreotide, procarbazine, suramin, photodynamic compoundssuch as methoxsalen and sodium porfimer, and proteasome inhibitors suchas bortezomib.

Molecular Targeted Therapy Agents Including:

functional therapeutic agents, e.g., gene therapy agents;

antisense therapy agents;

tyrosine kinase inhibitors such as erlotinib hydrochloride, gefitinib,imatinib mesylate, and semaxanib;

RAF inhibitors such as sorafenib;

gene expression modulators such as the retinoids and rexinoids, forexample adapalene, bexarotene, trans-retinoic acid, 9-cis-retinoic acid,and N-(4-hydroxyphenyl)retinamide; phenotype-directed therapy agents,including monoclonal antibodies such as alemtuzumab, bevacizumab,cetuximab, ibritumomab tiuxetan, rituximab, and trastuzumab;

immunotoxins such as emtansine, radioimmunoconjugates such asI-tositumobab, and cancer vaccines.

Biologic Therapy Agents Including:

interferons such as interferon-[alpha]2a and interferon-[alpha]2b, and

interleukins such as aidesleukin, denileukin diftitox, and oprelvekin.Axl inhibiting agents including1-(6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N3-((7-(S)-pyrrolidin-1-yl)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine(BGB324/R428), CH5451098 (Roche) and Axl inhibitors described inPCT/US07/089177, PCT/US2010/021275 and PCT/EP2011/004451, incorporatedherein by reference.

In addition to these agents intended to act against cancer cells,anticancer therapies include the use of protective or adjunctive agents,including:

cytoprotective agents such as amifostine, and dexrazoxane;

phosphonates such as pamidronate and zoledronic acid; and

stimulating factors such as epoetin, darbeopetin, filgrastim,PEG-filgrastim, and sargramostim.

Many combination chemotherapeutic regimens are known to the art, such ascombinations of carboplatin/paclitaxel, capecitabine/docetaxel,fluorauracil/evamisole, fluorauracil/leucovorin,methotrexate/leucovorin, and trastuzumab/paclitaxel, alone or in furthercombination with carboplatin, and the like.

A particularly preferred class of agent for use in combination with theanti-Axl antibodies disclosed herein are Immune Checkpoint Modulators(ICMs) such as Immune Checkpoint Inhibitors (ICIs).

Immune checkpoints, which are inhibitory pathways in the immune system,may be co-opted by tumours to induce immune resistance. The use ofantibodies to block or modulate immune checkpoints, including T-cellstimulatory and inhibitory receptors and dendritic cell stimulatoryreceptors, and thus to reduce or reverse the immune resistance of thecancer, is thus an important avenue in cancer research.

T-cell stimulatory receptors that may be modulated through the use ofimmune checkpoint modulating antibodies include CD28, ICOS, 4-1BB, OX40,GITR, CD27, TWEAKR, HVEM and TIM-1. T-cell inhibitory receptors thatmaybe modulated through the use of immune checkpoint modulatingantibodies include PD-L1, CTLA-4, PD-1, BTLA, TIM-3, VISTA, LAG-3 andTIGIT. Dendritic cell stimulatory receptors that may be modulatedthrough the use of immune checkpoint modulating antibodies include CD40and 4-1BB.

Thus ICMs suitable for use in combination with the anti-Axl antibodiesdisclosed herein include the immune checkpoint modulating, orinhibiting, antibodies of which there are many known in the art.Particularly suitable immune checkpoint modulating antibodies include:

-   -   CTLA-4 targeting antibodies, including Ipilimumab and        Tremelimumab.    -   PD-1 targeting antibodies, including Pembrolizumab, Mivolumab        and AMP-514/MEDI0680.    -   BD-L1 targeting antibodies, including MPDL3280A, MEDI4736,        MSB0010718C and BMS-936559.    -   4-1 BB targeting antibodies, including Urelumab and PF-05082566.    -   OX-40 targeting antibodies, including MEDI6469, MEDI6383        (rOX40L) and MOXR0916.    -   GITR targeting antibodies, including TRX518.    -   CD27 targeting antibodies, including CDX-1127.    -   CD40 targeting antibodies, including CP-870,893.    -   LAG3 targeting antibodies, including BMS-986016.

Where a combination of ICM antibodies are used in conjunction with ananti-AXL antibody of the invention, all of the ICM antibodies used maytarget inhibitory receptors, all of the ICM antibodies used may targetstimulatory receptors, or a combination of inhibitory receptor andstimulatory receptor targeting ICM antibodies may be used.

The disclosure therefore provides an antibody that binds Axl, asdescribed herein, for use in treatment (of, for example, a proliferativedisease such as cancer), wherein the treatment further comprises one ormore immune checkpoint modulating antibodies. Likewise, there isprovided an antibody that binds Axl, as described herein, in themanufacture of a medicament for the treatment of a proliferative disease(such as cancer), wherein the treatment further comprises one or moreimmune checkpoint modulating antibodies. The antibodies may be selectedfrom Ipilimumab, Tremelimumab, Pembrolizumab, Mivolumab,AMP-514/MEDI0680, MPDL3280A, MEDI4736, MSB0010718C, BMS-936559,Urelumab, PF-05082566, MEDI6469, MEDI6383 (rOX40L), MOXR0916, TRX518,CDX-1127, CP-870,893 and BMS-986016. The cancer may be selected fromlung cancer, melanoma, breast cancer, ovarian cancer or carcinoma.

The compound of the invention may be administered before the one or moreimmune checkpoint modulating antibodies, simultaneously with the one ormore immune checkpoint modulating antibodies, or after the one or moreimmune checkpoint modulating antibodies.

Another particularly preferred class of agent for use in combinationwith the anti-Axl antibodies of the present invention are anti-tumourantibodies specific for a target other than Axl. Such antibodiessuitable for use in combination with the anti-Axl antibodies of thepresent invention are set out in the table below:

Examples of therapeutic mAbs Antigen Examples of raised against theseTumour types expressing category antigens targets antigen HaematopoieticCD20 Rituximab Non-Hodgkin's lymphoma differentiation Ibritumomabtiuxetan Lymphoma antigens and tositumomab CD30 Brentuximab vedotinHodgkin's lymphoma CD33 Gemtuzumab Acute myelogenous ozogamicinleukaemia CD52 Alemtuzumab Chronic lymphocytic leukaemia GlycoproteinsEpCAM IGN101 and Epithelial tumours (breast, expressed by adecatumumabcolon and lung) solid tumours CEA Labetuzumab Breast, colon and lungtumours gpA33 huA33 Colorectal carcinoma Mucins Pemtumomab and Breast,colon, lung and oregovomab ovarian tumours TAG-72 CC49 (minretumomab)Breast, colon and lung tumours CAIX cG250 Renal cell carcinoma PSMA J591Prostate carcinoma Folate-binding MOv18 and MORAb- Ovarian tumoursprotein 003 (farletuzumab) Glycolipids Gangliosides 3F8, ch14.18 and KW-Neuroectodermal tumours (such as GD2, 2871 and some epithelial tumoursGD3 and GM2) Carbohydrates Le^(y) hu3S193 and IgN311 Breast, colon, lungand prostate tumours Targets of anti- VEGF Bevacizumab Tumourvasculature angiogenic VEGFR IM-2C6 and CDP791 Epithelium-derived solidmAbs tumours Integrin αVβ3 Etaracizumab Tumour vasculature Integrin α5β1Volociximab Tumour vasculature Growth and EGFR Cetuximab, Glioma, lung,breast, colon, differentiation panitumumab, and head and neck tumourssignalling nimotuzumab and 806 ERBB2 Trastuzumab and Breast, colon,lung, ovarian pertuzumab and prostate tumours ERBB3 MM-121 Breast,colon, lung, ovarian and prostate, tumours MET AMG 102, METMAB Breast,ovary and lung and SCH 900105 tumours IGF1R AVE1642, IMC-A12, Glioma,lung, breast, head MK-0646, R1507 and and neck, prostate and CP 751871thyroid cancer EPHA3 KB004 and IIIA4 Lung, kidney and colon tumours,melanoma, glioma and haematological malignancies TRAILR1 Mapatumumab(HGS- Colon, lung and pancreas ETR1) tumours and haematologicalmalignancies TRAILR2 HGS-ETR2 and CS- 1008 RANKL Denosumab Prostatecancer and bone metastases Stromal and FAP Sibrotuzumab and F19 Colon,breast, lung, extracellular pancreas, and head and matrix antigens necktumours Tenascin 81C6 Glioma, breast and prostate tumours

Throughout the specification, preferably the methods described hereinare performed in vitro or ex vivo. Methods can also be performed invivo.

The present invention provides a method comprising causing or allowingbinding of an antibody as provided herein to Axl. As noted, such bindingmay take place in vivo, e.g. following administration of an antibody, ornucleic acid encoding an antibody, or it may take place in vitro, forexample in ELISA, Western blot analysis, immunocytochemistry,immunohistochemistry, immunoprecipitation or affinity chromatography.

The amount of antibody bound to Axl receptor may be determined.Quantitation may be related to the amount of the antigen in a testsample, which may be of diagnostic interest.

The reactivity of antibody in a sample may be determined by anyappropriate means. Radioimmunoassay (RIA) is one possibility.Radioactively labelled antigen is mixed with unlabelled antigen (thetest sample) and allowed to bind to the antibody. Bound antigen isphysically separated from unbound antigen and the amount of radioactiveantigen bound to the antibody determined. The more antigen there is inthe test sample the less radioactive antigen will bind to the antibody.A competitive binding assay may also be used with non-radioactiveantigen, using antigen or an analogue linked to a reporter molecule. Thereporter molecule may be a fluorochrome, phosphor or laser dye withspectrally isolated absorption or emission characteristics. Suitablefluorochromes include fluorescein, rhodamine, phycoerythrin and TexasRed. Suitable chromogenic dyes include diaminobenzidine.

Other reporters include macromolecular colloidal particles orparticulate material such as latex beads that are coloured, magnetic orparamagnetic, and biologically or chemically active agents that candirectly or indirectly cause detectable signals to be visually observed,electronically detected or otherwise recorded. These molecules may beenzymes which catalyse reactions that develop or change colours or causechanges in electrical properties, for example. They may be molecularlyexcitable, such that electronic transitions between energy states resultin characteristic spectral absorptions or emissions. They may includechemical entities used in conjunction with biosensors. Biotin/avidin orbiotin/streptavidin and alkaline phosphatase detection systems may beemployed.

The signals generated by individual antibody-reporter conjugates may beused to derive quantifiable absolute or relative data of the relevantantibody binding in samples (normal and test).

The present invention also provides the use of an antibody as above formeasuring antigen levels in a competition assay, that is to say a methodof measuring the level of antigen in a sample by employing an antibodyas provided by the present invention in a competition assay. This may bewhere the physical separation of bound from unbound antigen is notrequired. Linking a reporter molecule to the antibody so that a physicalor optical change occurs on binding is one possibility. The reportermolecule may directly or indirectly generate detectable, and preferablymeasurable, signals. The linkage of reporter molecules may be directlyor indirectly, covalently, e.g. via a peptide bond or non-covalently.Linkage via a peptide bond may be as a result of recombinant expressionof a gene fusion encoding antibody and reporter molecule.

The present invention also provides for measuring levels of antigendirectly, by employing an antibody according to the invention forexample in a biosensor system.

The mode of determining binding is not a feature of the presentinvention and those skilled in the art are able to choose a suitablemode according to their preference and general knowledge.

The present invention further extends to an antibody that competes forbinding to Axl with any antibody that both binds the antigen andcomprises an antibody variable domain (either VH or VL or both)including a CDR with amino acid substantially as set out herein or avariable domain with amino acid sequence substantially as set outherein. Competition between the antibodies may be assayed easily invitro, for example by tagging a specific reporter molecule to onebinding member that can be detected in the presence of other untaggedbinding member(s), to enable identification of antibodies that bind thesame epitope or an overlapping epitope. Competition may be determinedfor example using ELISA or flow cytometry. Alternatively, competingantibodies may be identified via surface plasmon resonase (SPR)technique using Biacore instrument, as described in Example 6.

In another method, to screen for antibodies that bind to the epitope onAxl bound by an antibody of interest (e.g, those that block binding ofthe 10C9 or 10G5 antibody to Axl), a routine cross-blocking assay suchas that described in Antibodies. A Laboratory Manual. Cold Spring HarborLaboratory. Ed Harlow and David Lane (1988), can be performed.

In testing for competition, a peptide fragment of the antigen may beemployed, especially a peptide including an epitope of interest. Apeptide having the epitope sequence plus one or more amino acids ateither end may be used. Such a peptide may be said to “consistessentially” of the specified sequence. Antibodies according to thepresent invention may be such that their binding for antigen isinhibited by a peptide with or including the sequence given. In testingfor this, a peptide with either sequence plus one or more amino acidsmay be used.

Antibodies that bind a specific peptide may be isolated for example froma phage display library by panning with the peptide(s).

The present invention further provides an isolated nucleic acid encodingan antibody of the present invention. Nucleic acid includes DNA and RNA.In a preferred aspect, the present invention provides a nucleic acidthat codes for a CDR, VH or VL domain of the invention as defined above.

The present invention also provides constructs in the form of plasmids,vectors, transcription or expression cassettes that comprise at leastone polynucleotide as above.

The present invention also provides a recombinant host cell thatcomprises one or more constructs as above. A nucleic acid encoding anyCDR, VH or VL domain, or antibody as provided, itself forms an aspect ofthe present invention, as does a method of production of the encodedproduct, which method comprises expression from encoding nucleic acidtherefor. Expression may conveniently be achieved by culturing underappropriate conditions recombinant host cells containing the nucleicacid. Following production by expression, a VH or VL domain, or antibodymay be isolated and/or purified using any suitable technique known inthe art.

Antibodies, VH and/or VL domains, and encoding nucleic acid moleculesand vectors according to the present invention may be provided isolatedand/or purified, e.g. from their natural environment, in substantiallypure or homogeneous form, or, in the case of nucleic acid, free orsubstantially free of nucleic acid or genes of an origin other than thesequence encoding a polypeptide with the required function. Nucleic acidaccording to the present invention may comprise DNA or RNA and may bewholly or partially synthetic. Reference to a nucleotide sequence as setout herein encompasses a DNA molecule with the specified sequence, andencompasses a RNA molecule with the specified sequence in which U issubstituted for T, unless context requires otherwise.

Systems for cloning and expression of a polypeptide in a variety ofdifferent host cells are well known. Suitable host cells includebacteria, mammalian cells, yeast, baculovirus, and insect cell systems.Mammalian cell lines available in the art for expression of aheterologous polypeptide include Chinese hamster ovary cells (CHO), HeLacells, baby hamster kidney (BHK) cells, NS0 and SP2/0 mouse myelomacells, YB2/0 rat myeloma cells, human cell lines HEK-293 and PER.C6 andmany others. A common, preferred bacterial host is E. coli.

The expression of antibodies and antibody fragments in prokaryotic cellssuch as E. coli is well established in the art. For a review, see forexample PIückthun, A. Bio/Technology 9: 545-551 (1991). Expression ineukaryotic cells in culture is also available to those skilled in theart as an option for production of an antibody, see for reviews, forexample Ref, M. E. (1993) Curr. Opinion Biotech. 4: 573-576; Trill J. J.et al. (1995) Curr. Opinion Biotech 6: 553-560.

Suitable vectors can be chosen or constructed, containing appropriateregulatory sequences, including promoter sequences, terminatorsequences, polyadenylation sequences, enhancer sequences, marker genesand other sequences as appropriate. Vectors may be plasmids, viral e.g.phage, or phagemid, as appropriate (Sambrook and Russell, 2001,Molecular Cloning: a Laboratory Manual: 3^(rd) edition, Cold SpringHarbor Laboratory Press). Many known techniques and protocols formanipulation of nucleic acid, for example in preparation of nucleic acidconstructs, mutagenesis, sequencing, introduction of DNA into cells andgene expression, and analysis of proteins, are described in detail inCurrent Protocols in Molecular Biology, Second Edition, Ausubel et al.eds., John Wiley & Sons, 1992.

Thus, a further aspect of the present invention provides a host cellcontaining nucleic acid as disclosed herein. A still further aspectprovides a method comprising introducing such nucleic acid into a hostcell. The introduction may employ any available technique. Foreukaryotic cells, suitable techniques may include calcium phosphatetransfection, DEAE-Dextran, electroporation, liposome-mediatedtransfection and transduction using retrovirus or other virus, e.g.vaccinia or, for insect cells, baculovirus. For bacterial cells,suitable techniques may include calcium chloride transformation,electroporation and transfection using bacteriophage.

The introduction may be followed by causing or allowing expression fromthe nucleic acid, e.g. by culturing host cells under conditions forexpression of the gene.

In one embodiment, the nucleic acid of the invention is integrated intothe genome (e.g. chromosome) of the host cell. Integration may bepromoted by inclusion of sequences that promote recombination with thegenome, in accordance with standard techniques.

The present invention also provides a method that comprises using aconstruct as stated above in an expression system in order to express anantibody or polypeptide as above.

Aspects and embodiments of the present invention will now be illustratedby way of example with reference to the following experimentation.

All documents cited anywhere in this specification are incorporated byreference.

Statements of Invention—10C9 Antibody

-   -   The following paragraphs describe a number of specifically        envisioned embodiments and combinations of the present        invention.

1. An antibody that binds Axl and which comprises:

-   -   an antibody VH domain selected from the group consisting of the        10C9 VH domain (SEQ ID NO.3) and a VH domain comprising a VH        CDR3 with the amino acid sequence of SEQ ID NO.7 and optionally        one or more VH CDR's with an amino acid sequence selected from        SEQ ID NO.6 and SEQ ID NO.5; and/or    -   an antibody VL domain selected from the group consisting of the        10C9 VL domain (SEQ ID NO. 4) and a VL domain comprising one or        more VL CDR's with an amino acid sequence selected from SEQ ID        NO.8, SEQ ID NO.9 and SEQ ID NO.10.

2. An antibody according to paragraph 1 comprising an antibody VH domaincomprising the VH CDR's with the amino acid sequences of SEQ ID NO.5,SEQ ID NO.6 and SEQ ID NO.7, which antibody competes for binding to Axlwith an Axl binding domain of an antibody comprising the 10C9 VH domain(SEQ ID NO. 3) and the 10C9 VL domain (SEQ ID NO. 4).

3. An antibody according to paragraph 1 or paragraph 2 comprising the10C9 VH domain (SEQ ID NO. 3).

4. An antibody according to paragraph 3 comprising the 10C9 VL domain(SEQ ID NO. 4).

5. A variant of an antibody according to any one of paragraphs 1 to 4,wherein the variant comprises one or more amino acid sequencealterations in one or more framework regions and/or one or more CDRs.

6. An antibody according to any one of paragraphs 1 to 5 that binds Axlwith affinity equal to or better than the affinity of an Axlantigen-binding site formed by the 10C9 VH domain (SEQ ID NO. 3) and the10C9 VL domain (SEQ ID NO. 4), the affinity of the antibody and theaffinity of the antigen-binding site being as determined under the sameconditions.

7. An antibody according to any one of paragraphs 1 to 6 that comprisesan scFv antibody molecule.

8. An antibody according to any one of paragraphs 1 to 6 that comprisesan antibody constant region.

9. An antibody according to paragraph 8 that comprises a whole antibody.

10. An antibody according to any one of paragraphs 1 to 9 that comprisesadditional amino acids providing a further functional characteristic inaddition to the ability to bind antigen.

11. An antibody according to any one of paragraphs 1 to 10 that bindsAxl with a K_(D) no greater than 2×10⁻¹⁰ M.

12. An antibody according to any one of paragraphs 1 to 11 that bindsAxl with a k_(on) no lower than 1.5×10⁶ M⁻¹s⁻¹.

13. An antibody according to any one of paragraphs 1 to 12 wherein theAxl is human Axl.

14. An antibody according to any one of paragraphs 1 to 13 thatspecifically binds primate Axl.

15. An antibody according to any one of paragraphs 1 to 14 that:

-   -   (i) binds murine Axl with a K_(D) greater than 10⁻³ M;    -   (ii) binds human Mer with a K_(D) greater than 10⁻³ M; and/or    -   (iii) binds human Tyro3 with a K_(D) greater than 10⁻³ M.

16. An antibody according to any one of paragraphs 1 to 15 that inhibitsthe binding of Axl to Gas6.

17. An antibody according to any one of paragraphs 1 to 16 thatdown-regulates expression of the Axl receptor.

18. An antibody according to paragraph 17, wherein the antibody reducesAxl receptor expression to less than 50% of the level observed in anotherwise identically treated sample that is not contacted with to theantibody.

19. An antibody according to either one of paragraphs 17 or 18, whereinthe downregulation of Axl receptor expression is observed within 12hours of contacting the sample with the antibody.

20. An antibody according to any one of paragraphs 17 to 19, wherein thedown regulation of Axl receptor expression persists for at least 24hours following contacting the sample with the antibody.

21. An antibody according to any one of paragraphs 1 to 20 thatincreases the rate of Axl receptor internalization.

22. An antibody according to any one of paragraphs 1 to 21 that inhibitsAxl activity.

23. An antibody according to paragraph 22, wherein the antibody inhibitsAxl receptor downstream signalling.

24. An antibody according to either one of paragraphs 22 or 23 whereinthe phosphorylation of Aid at Serine 473 in a sample contacted with theantibody of the invention is less than 50% of the level observed in anotherwise identically treated sample that is not contacted with theantibody

25. An antibody according to any one of paragraphs 1 to 24 thatincreases the rate of cell death.

26. An antibody according to any one of paragraphs 1 to 25 that inhibitstumour growth.

27. An antibody according to any one of paragraphs 1 to 26 that isconjugated to a detectable label, enzyme, or toxin, optionally via apeptidyl bond or linker.

28. An antibody according to paragraph 27 wherein the toxin is selectedfrom the group comprising MMAE and MMAF.

29. An antibody according to paragraph 27 wherein the detectable labelis FITC.

30. An antibody according to any one of paragraphs 1 to 29 binds to theepitope bound by the 10C9 antibody obtainable from the hybridomaUT-10C9-B9.

31. An antibody that binds to the epitope bound by the 10C9 antibodyobtainable from the hybridoma UT-10C9-B9.

32. The antibody according to paragraph 31 that inhibits the binding ofAxl to its ligand Gas6.

33. The antibody according to either one of paragraphs 31 or 32 thatdownregulates Axl expression, inhibits Axl receptor signalling, and/orinhibits tumour growth.

34. An 10C9 antibody obtainable from the hybridoma UT-10C9-B9.

35. An isolated nucleic acid that comprises a nucleotide sequenceencoding an antibody or antibody VH or VL domain of an antibodyaccording to any one of paragraphs 1 to 26.

36. A host cell transformed with nucleic acid according to paragraph 35.

37. A method of producing an antibody or antibody VH or VL domain, themethod comprising culturing host cells according to paragraph 36 underconditions for production of said antibody or antibody VH or VL domain.

38. A method according to paragraph 37 further comprising isolatingand/or purifying said antibody or antibody VH or VL variable domain.

39. A method according to paragraph 37 or paragraph 38 furthercomprising formulating the antibody or antibody VH or VL variable domaininto a composition including at least one additional component.

40. A method of obtaining an antibody that binds Axl, the methodcomprising

-   -   providing by way of addition, deletion, substitution or        insertion of one or more amino acids in the amino acid sequence        of the 10C9 VH domain (SEQ ID NO. 3) one or more VH domains each        of which is an amino acid sequence variant of the 10C9 VH        domain, optionally combining one or more VH domain amino acid        sequence variants thus provided with one or more VL domains to        provide one or more VH/VL combinations; and/or    -   providing by way of addition, deletion, substitution or        insertion of one or more amino acids in the amino acid sequence        of the 10C9 VL domain (SEQ ID NO. 4) a VL domain that is an        amino acid sequence variant of the 10C9 VL domain, and combining        one or more VL domain amino acid sequence variants thus provided        with one or more VH domains to provide one or more VH/VL domain        combinations;

and

-   -   testing the VH domain amino acid sequence variants or VH/VL        combination or combinations for to identify a antibody that        binds Axl.

41. A method of obtaining an antibody that binds Axl, which methodcomprises:

-   -   providing starting nucleic acids encoding one or more VH domains        that either comprise a CDR3 to be replaced or lack a CDR3        encoding region, and combining said starting nucleic acid with a        donor nucleic acid encoding the VH CDR3 amino acid sequence of        SEQ ID NO.7 such that said donor nucleic acid is inserted into        the CDR3 region in the starting nucleic acid, so as to provide        product nucleic acids encoding VH domains; or    -   providing starting nucleic acids encoding one or more VL domains        that either comprise a CDR3 to be replaced or lack a CDR3        encoding region, and combining said starting nucleic acid with a        donor nucleic acid encoding the VL CDR3 amino acid sequence of        SEQ ID NO.10 such that said donor nucleic acid is inserted into        the CDR3 region in the starting nucleic acid, so as to provide        product nucleic acids encoding VL domains;    -   expressing the nucleic acids of said product nucleic acids        encoding VH domains and optionally combining the VH domains thus        produced with one or more VL domains to provide VH/VL        combinations, and/or expressing the nucleic acids of said        product nucleic acids encoding VL domains and combining the VL        domains thus produced with one or more VH domains to provide        VH/VL combinations;    -   selecting an antibody comprising a VH domain or a VH/VL        combination that binds Axl; and    -   recovering said antibody that binds Axl and/or nucleic acid        encoding the antibody that binds Axl.

42. A method according to paragraph 41 or paragraph 41 wherein theantibody that binds Axl is an antibody fragment comprising a VH domainand a VL domain.

43. A method according to paragraph 42 wherein the antibody fragment isan scFv antibody molecule.

44. A method according to paragraph 42 wherein the antibody fragment isan Fab antibody molecule.

45. A method according to paragraph 43 or paragraph 44 furthercomprising providing the VH domain and/or the VL domain of the antibodyfragment in a whole antibody.

46. A method according to any one of paragraphs 37 to 45 furthercomprising formulating the antibody that binds Axl or an antibody VH orVL variable domain of the antibody that binds Axl into a compositionincluding at least one additional component.

47. A method according to any one of paragraphs 37 to 46 furthercomprising binding an antibody that binds Axl to Axl or a fragment ofAxl.

48. A method comprising binding an antibody that binds Axl according toany one of paragraphs 1 to 29 to Axl or a fragment of Axl.

49. A method according to paragraph 47 or paragraph 48 wherein saidbinding takes place in vitro.

50. A method according to any one of paragraphs 47 to 49 comprisingdetermining the amount of binding of antibody to Axl or a fragment ofAxl.

51. A method according to any one of paragraphs 37 to 46 furthercomprising use of the antibody in the manufacture of a medicament fortreatment of a disease or disorder characterised by overexpression ofAxl.

52. A composition comprising an antibody according to any one ofparagraphs 1 to 26, or an immunoconjugate thereof, in conjunction with apharmaceutically acceptable excipient.

53. The composition according to paragraph 52, further comprising anImmune Checkpoint Modulator, and/or an anti-tumour antibody specific fora target other than Axl.

54. The composition according to paragraph 53, wherein the ImmuneCheckpoint Modulator is an antibody, such as Ipilimumab, Tremelimumab,Pembrolizumab, Mivolumab, AMP-514/MEDI0680, MPDL3280A, MEDI4736,MSB0010718C, BMS-936559, Urelumab, PF-05082566, MEDI6469, MEDI6383(rOX40L), MOXR0916, TRX518, CDX-1127, CP-870,893 or BMS-986016.

55. The composition according to paragraph 53, wherein the anti-tumourantibody specific for a target other than Axl is selected from the groupconsisting of Rituximab, Ibritumomab tiuxetan, tositumomab, Brentuximabvedotin, Gemtuzumab ozogamicin, Alemtuzumab, IGN101, adecatumumab,Labetuzumab, huA33, Pemtumomab, oregovomab, CC49 (minretumomab), cG250,J591, MOv18, MORAb-003 (farletuzumab), 3F8, ch14.18, KW-2871, hu3S193,IgN311, Bevacizumab, IM-2C6, CDP791, Etaracizumab, Volociximab,Cetuximab, panitumumab, nimotuzumab 806, Trastuzumab, pertuzumab,MM-121, AMG 102, METMAB, SCH 900105, AVE1642, IMC-A12, MK-0646, R1507,CP 751871, KB004, IIIA4, Mapatumumab (HGS-ETR1), HGS-ETR2,CS-1008,Denosumab, Sibrotuzumab, F19, 81C6.

56. An antibody according to any one of paragraphs 1 to 29, or thecomposition according to any one of paragraphs 52 to 55, for use in amethod of treatment.

57. An antibody or composition according to paragraph 56 for use in amethod of treating a proliferative disease.

58. An antibody or composition according to paragraph 57 where theproliferative disease is cancer.

59. An antibody or composition according to paragraph 58 where thecancer is metastatic cancer.

60. Use of an antibody according to any one of paragraphs 1 to 29, orthe composition according to any one of paragraphs 52 to 55, in themanufacture of a medicament for treatment of a disease or disordercharacterised by overexpression of Axl.

61. A method of treatment of a disease or disorder characterised byoverexpression of Axl, the method comprising administering an antibodyaccording to any one of paragraphs 1 to 29, or the composition accordingto any one of paragraphs 52 to 55, to a patient with the disease ordisorder or at risk of developing the disease or disorder.

62. An antibody according to any one of paragraphs 56 to 59, or methodof claim 61, wherein the method of treatment comprises administering theantibody according to any one of paragraphs 1 to 29, or the compositionaccording to any one of paragraphs 52 to 55, in combination with anImmune Checkpoint Modulator and/or an anti-tumour antibody specific fora target other than Axl.

63. A method according to paragraph 61 wherein the antibody directs thedelivery of a pharmaceutical composition to target metastatic cancercells.

64. Use of an antibody according to any one of paragraphs 1 to 29 andone or more reagents that allow determination of the binding of saidantibody to metastatic cancer cells, in the manufacture of a diagnosticagent for the detection of a disease or disorder characterised byoverexpression of Axl.

65. A method of diagnosis of a disease or disorder characterised byoverexpression of Axl, the method comprising administering an antibodyaccording to any one of paragraphs 1 to 29, or the composition accordingto any one of paragraphs 52 to 55, and one or more reagents that allowdetermination of the binding of said antibody to metastatic cancercells, to a patient with the disease or disorder or at risk ofdeveloping the disease or disorder.

66. A diagnostic kit comprising an antibody according to any one ofparagraphs 1 to 29 and one or more reagents that allow determination ofthe binding of said member to metastatic cancer cells.

67. A kit comprising an antibody according to any one of paragraphs 1 to29, or the composition according to any one of paragraphs 52 to 55.

68. A pharmaceutical composition comprising as active principle anantibody according to paragraphs 1-29 in an effective amount, inconjunction with a pharmaceutically acceptable excipient.

Statements of Invention—10G5 Antibody

The following paragraphs describe a number of specifically envisionedembodiments and combinations of the present invention.

1a. An antibody that binds Axl and which comprises:

-   -   an antibody VH domain selected from the group consisting of the        10G5 VH domain (SEQ ID NO.21) and a VH domain comprising a VH        CDR3 with the amino acid sequence of SEQ ID NO.25 and optionally        one or more VH CDR's with an amino acid sequence selected from        SEQ ID NO.24 and SEQ ID NO.23; and/or    -   an antibody VL domain selected from the group consisting of the        10G5 VL domain (SEQ ID NO. 22) and a VL domain comprising one or        more VL CDR's with an amino acid sequence selected from SEQ ID        NO.26, SEQ ID NO.27 and SEQ ID NO.28.

1b. An antibody that binds Axl and which comprises:

-   -   an antibody VH domain selected from the group consisting of the        10G5(Q1E) VH domain (SEQ ID NO45) and a VH domain comprising a        VH CDR3 with the amino acid sequence of SEQ ID NO.25 and        optionally one or more VH CDR's with an amino acid sequence        selected from SEQ ID NO.24 and SEQ ID NO.23; and/or    -   an antibody VL domain selected from the group consisting of the        10G5 VL domain (SEQ ID NO. 22) and a VL domain comprising one or        more VL CDR's with an amino acid sequence selected from SEQ ID        NO.26, SEQ ID NO.27 and SEQ ID NO.28.

2a. An antibody according to paragraph 1a or 1 b comprising an antibodyVH domain comprising the VH CDR's with the amino acid sequences of SEQID NO.23, SEQ ID NO.24 and SEQ ID NO.25, which antibody competes forbinding to Axl with an Axl binding domain of an antibody comprising the10G5 VH domain (SEQ ID NO. 21) and the 10G5 VL domain (SEQ ID NO. 22).

3a. An antibody according to paragraph 1a or paragraph 2a comprising the10G5 VH domain (SEQ ID NO. 21).

3b. An antibody according to paragraph 1b or paragraph 2a comprising the10G5(Q1E) VH domain (SEQ ID NO. 45).

4a. An antibody according to paragraph 3a comprising the 10G5 VL domain(SEQ ID NO. 22)

5a. A variant of an antibody according to any one of paragraphs 1a to4a, wherein the variant comprises one or more amino acid sequencealterations in one or more framework regions and/or one or more CDRs.

6a. An antibody according to any one of paragraphs 1a to 5a that bindsAxl with affinity equal to or better than the affinity of an Axlantigen-binding site formed by the 10G5 VH domain (SEQ ID NO. 21) andthe 10G5 VL domain (SEQ ID NO. 22), the affinity of the antibody and theaffinity of the antigen-binding site being as determined under the sameconditions.

7a. An antibody according to any one of paragraphs 1a to 6a thatcomprises an scFv antibody molecule.

a8. An antibody according to any one of paragraphs 1a to 6a thatcomprises an antibody constant region.

9a. An antibody according to paragraph 8a that comprises a wholeantibody.

10a An antibody according to any one of paragraphs 1a to 9a thatcomprises additional amino acids providing a further functionalcharacteristic in addition to the ability to bind antigen.

11a. An antibody according to any one of paragraphs 1a to 10a that bindsAxl with a K_(D) no greater than 6×10⁻¹⁰ M.

12a. An antibody according to any one of paragraphs 1a to 11a that bindsAxl with a k_(on) no lower than 8×10⁵ M⁻¹s⁻¹.

13a. An antibody according to any one of paragraphs 1a to 12a whereinthe Axl is human Axl.

14a. An antibody according to any one of paragraphs 1a to 13a thatspecifically binds primate Axl.

15a. An antibody according to any one of paragraphs 1a to 14a that:

-   -   (i) binds murine Axl with a K_(D) greater than 10⁻³ M;    -   (ii) binds human Mer with a K_(D) greater than 10⁻³ M; and/or    -   (iii) binds human Tyro3 with a K greater than 10⁻³ M.

16a. An antibody according to any one of paragraphs 1a to 15a thatinhibits the binding of Axl to Gas6.

17a. An antibody according to any one of paragraphs 1a to 16a thatdownregulates expression of the Axl receptor.

18a. An antibody according to paragraph 17a, wherein the antibodyreduces Axl receptor expression to less than 50% of the level observedin an otherwise identically treated sample that is not contacted with tothe antibody.

19a. An antibody according to either one of paragraphs 17a or 18a,wherein the downregulation of Axl receptor expression is observed within12 hours of contacting the sample with the antibody.

20a. An antibody according to any one of paragraphs 17a to 19a, whereinthe down regulation of Axl receptor expression persists for at least 24hours following contacting the sample with the antibody.

21a. An antibody according to any one of paragraphs 1a to 20a thatincreases the rate of Axl receptor internalization.

22a. An antibody according to any one of paragraphs 1a to 21a thatinhibits Axl activity.

23a. An antibody according to paragraph 22a, wherein the antibodyinhibits Axl receptor downstream signalling.

24a. An antibody according to either one of paragraphs 22a or 23awherein the phosphorylation of Aid at Serine 473 in a sample contactedwith the antibody of the invention is less than 50% of the levelobserved in an otherwise identically treated sample that is notcontacted with the antibody

25a. An antibody according to any one of paragraphs 1a to 24a thatincreases the rate of cell death.

26a. An antibody according to any one of paragraphs 1a to 25a thatinhibits tumour growth.

27a. An antibody according to any one of paragraphs 1a to 26a that isconjugated to a detectable label, enzyme, or toxin, optionally via apeptidyl bond or linker.

28a. An antibody according to paragraph 27a wherein the toxin isselected from the group comprising MMAE and MMAF.

29a. An antibody according to paragraph 27a wherein the detectable labelis FITC.

30a. An antibody according to any one of paragraphs 1a to 29a binds tothe epitope bound by the 10G5 antibody obtainable from the hybridomaWR-10G5-E5.

31a. An antibody that binds to the epitope bound by the 10G5 antibodyobtainable from the hybridoma WR-10G5-E5.

32a. The antibody according to paragraph 31a that inhibits the bindingof Axl to its ligand Gas6.

33a. The antibody according to either one of paragraphs 31a or 32a thatdownregulates Axl expression, inhibits Axl receptor signalling, and/orinhibits tumour growth.

34a. An 10G5 antibody obtainable from the hybridoma WR-10G5-E5.

35a. An isolated nucleic acid that comprises a nucleotide sequenceencoding an antibody or antibody VH or VL domain of an antibodyaccording to any one of paragraphs 1a to 26a.

36a. A host cell transformed with nucleic acid according to paragraph35a.

37a. A method of producing an antibody or antibody VH or VL domain, themethod comprising culturing host cells according to paragraph 36a underconditions for production of said antibody or antibody VH or VL domain.

38a. A method according to paragraph 37a further comprising isolatingand/or purifying said antibody or antibody VH or VL variable domain.

39a. A method according to paragraph 37a or paragraph 38a furthercomprising formulating the antibody or antibody VH or VL variable domaininto a composition including at least one additional component.

40a. A method of obtaining an antibody that binds Axl, the methodcomprising

-   -   providing by way of addition, deletion, substitution or        insertion of one or more amino acids in the amino acid sequence        of the 10G5 VH domain (SEQ ID NO. 3) one or more VH domains each        of which is an amino acid sequence variant of the 10G5 VH        domain, optionally combining one or more VH domain amino acid        sequence variants thus provided with one or more VL domains to        provide one or more VH/VL combinations; and/or    -   providing by way of addition, deletion, substitution or        insertion of one or more amino acids in the amino acid sequence        of the 10G5 VL domain (SEQ ID NO. 4) a VL domain which is an        amino acid sequence variant of the 10G5 VL domain, and combining        one or more VL domain amino acid sequence variants thus provided        with one or more VH domains to provide one or more VH/VL domain        combinations;

and

-   -   testing the VH domain amino acid sequence variants or VH/VL        combination or combinations for to identify a antibody that        binds Axl.

41a. A method of obtaining an antibody that binds Axl, which methodcomprises:

-   -   providing starting nucleic acids encoding one or more VH domains        that either comprise a CDR3 to be replaced or lack a CDR3        encoding region, and combining said starting nucleic acid with a        donor nucleic acid encoding the VH CDR3 amino acid sequence of        SEQ ID NO.7 such that said donor nucleic acid is inserted into        the CDR3 region in the starting nucleic acid, so as to provide        product nucleic acids encoding VH domains; or    -   providing starting nucleic acids encoding one or more VL domains        that either comprise a CDR3 to be replaced or lack a CDR3        encoding region, and combining said starting nucleic acid with a        donor nucleic acid encoding the VL CDR3 amino acid sequence of        SEQ ID NO.10 such that said donor nucleic acid is inserted into        the CDR3 region in the starting nucleic acid, so as to provide        product nucleic acids encoding VL domains;    -   expressing the nucleic acids of said product nucleic acids        encoding VH domains and optionally combining the VH domains thus        produced with one or more VL domains to provide VH/VL        combinations, and/or expressing the nucleic acids of said        product nucleic acids encoding VL domains and combining the VL        domains thus produced with one or more VH domains to provide        VH/VL combinations;    -   selecting an antibody comprising a VH domain or a VH/VL        combination that binds Axl; and    -   recovering said antibody that binds Axl and/or nucleic acid        encoding the antibody that binds Axl.

42a. A method according to paragraph 41a or paragraph 41a wherein theantibody that binds Axl is an antibody fragment comprising a VH domainand a VL domain.

43a. A method according to paragraph 42a wherein the antibody fragmentis an scFv antibody molecule.

44a. A method according to paragraph 42a wherein the antibody fragmentis an Fab antibody molecule.

45a. A method according to paragraph 43a or paragraph 44a furthercomprising providing the VH domain and/or the VL domain of the antibodyfragment in a whole antibody.

46a. A method according to any one of paragraphs 37a to 45a furthercomprising formulating the antibody that binds Axl or an antibody VH orVL variable domain of the antibody that binds Axl into a compositionincluding at least one additional component.

47a. A method according to any one of paragraphs 37a to 46a furthercomprising binding an antibody that binds Axl to Axl or a fragment ofAxl.

48a. A method comprising binding an antibody that binds Axl according toany one of paragraphs 1a to 29a to Axl or a fragment of Axl.

49a. A method according to paragraph 47a or paragraph 48a wherein saidbinding takes place in vitro.

50a. A method according to any one of paragraphs 47a to 49a comprisingdetermining the amount of binding of antibody to Axl or a fragment ofAxl.

51a. A method according to any one of paragraphs 37a to 46a furthercomprising use of the antibody in the manufacture of a medicament fortreatment of a disease or disorder characterised by overexpression ofAxl.

52a. A composition comprising an antibody according to any one ofparagraphs 1a to 26a, or an immunoconjugate thereof, in conjunction witha pharmaceutically acceptable excipient.

53a. The composition according to paragraph 52a, further comprising anImmune Checkpoint Modulator, and/or an anti-tumour antibody specific fora target other than Axl.

54a. The composition according to paragraph 53a, wherein the ImmuneCheckpoint Modulator is an antibody, such as Ipilimumab, Tremelimumab,Pembrolizumab, Mivolumab, AMP-514/MEDI0680, MPDL3280A, MEDI4736,MSB0010718C, BMS-936559, Urelumab, PF-05082566, MEDI6469, MEDI6383(rOX40L), MOXR0916, TRX518, CDX-1127, CP-870,893 or BMS-986016.

55a. The composition according to paragraph 53a, wherein the anti-tumourantibody specific for a target other than Axl is selected from the groupconsisting of Rituximab, Ibritumomab tiuxetan, tositumomab, Brentuximabvedotin, Gemtuzumab ozogamicin, Alemtuzumab, IGN101, adecatumumab,Labetuzumab, huA33, Pemtumomab, oregovomab, CC49 (minretumomab), cG250,J591, MOv18, MORAb-003 (farletuzumab), 3F8, ch14.18, KW-2871, hu3S193,IgN311, Bevacizumab, IM-2C6, CDP791, Etaracizumab, Volociximab,Cetuximab, panitumumab, nimotuzumab 806, Trastuzumab, pertuzumab,MM-121, AMG 102, METMAB, SCH 900105, AVE1642, IMC-A12, MK-0646, R1507,CP 751871, KB004, IIIA4, Mapatumumab (HGS-ETR1), HGS-ETR2,CS-1008,Denosumab, Sibrotuzumab, F19, 81C6.

56a. An antibody according to any one of paragraphs 1a to 29a, or thecomposition according to any one of paragraphs 52a to 55a, for use in amethod of treatment.

57a. An antibody or composition according to paragraph 56a for use in amethod of treating a proliferative disease.

58a. An antibody or composition according to paragraph 57a where theproliferative disease is cancer.

59a. An antibody or composition according to paragraph 58a where thecancer is metastatic cancer.

60a. Use of an antibody according to any one of paragraphs 1a to 29a, orthe composition according to any one of paragraphs 52a to 55a, in themanufacture of a medicament for treatment of a disease or disordercharacterised by overexpression of Axl.

61a. A method of treatment of a disease or disorder characterised byoverexpression of Axl, the method comprising administering an antibodyaccording to any one of paragraphs 1a to 29a, or the compositionaccording to any one of paragraphs 52a to 55a, to a patient with thedisease or disorder or at risk of developing the disease or disorder.

62a. An antibody according to any one of paragraphs 56a to 59a, ormethod of claim 61a, wherein the method of treatment comprisesadministering the antibody according to any one of paragraphs 1a to 29a,or the composition according to any one of paragraphs 52a to 55a, incombination with an Immune Checkpoint Modulator and/or an anti-tumourantibody specific for a target other than Axl.

63a. A method according to paragraph 61a wherein the antibody directsthe delivery of a pharmaceutical composition to target metastatic cancercells.

64a. Use of an antibody according to any one of paragraphs 1a to 29a andone or more reagents that allow determination of the binding of saidantibody to metastatic cancer cells, in the manufacture of a diagnosticagent for the detection of a disease or disorder characterised byoverexpression of Axl.

65a. A method of diagnosis of a disease or disorder characterised byoverexpression of Axl, the method comprising administering an antibodyaccording to any one of paragraphs 1 to 29a, or the compositionaccording to any one of paragraphs 52a to 55a, and one or more reagentsthat allow determination of the binding of said antibody to metastaticcancer cells, to a patient with the disease or disorder or at risk ofdeveloping the disease or disorder.

66a. A diagnostic kit comprising an antibody according to any one ofparagraphs 1a to 29a and one or more reagents that allow determinationof the binding of said member to metastatic cancer cells.

67a. A kit comprising an antibody according to any one of paragraphs 1ato 29a, or the composition according to any one of paragraphs 52a to55a.

68a. A pharmaceutical composition comprising as active principle anantibody according to paragraphs 1-29a in an effective amount, inconjunction with a pharmaceutically acceptable excipient.

EXAMPLES Example 1: Generation of Mouse Anti-Axl Monoclonal Antibodies

Monoclonal antibodies (MAb) against human Axl receptor were generated byDNA immunization of immunocompetent NMRI mice (Charles River) with aplasmid encoding a full-length human Axl fused to C-terminal Mycepitope.

Spleen cells from mice showing presence of rhAxl-specific antibodies inthe blood were used for fusion with mouse myeloma cells according tostandard protocols. The cells were cultured in plates (10⁵ cells perwell) with hypoxanthine-aminopterin-thymidine (HAT) medium for hybridomaselection. After twelve days of selection, the supernatants of 14generated hybridomas were harvested and tested for Axl binding inenzyme-linked immunosorbent assay (ELISA) and flow cytometry. Threepositive clones, showing the highest antigen-binding activity after thesecond round of subcloning by limited dilution, were expanded for largescale antibody production in vitro. The MAbs were purified from the cellculture supernatants by Protein G affinity chromatography.

The antibody clones 10C9 and 10G5 showing specific binding to Axl⁺ cellsin flow cytometry were selected for further characterization.

For flow cytometry, the adherent cells in culture were washed with PBS,detached by trypsin (0.25%) treatment for 1 min and hitting culture dishfor full detachment. Trypsin was quenched by adding into the tissueflask the complete medium followed by washing the cells with PBS. Duringthe washing steps, the cells were collected by centrifugation at 200 gfor 5 min. The antibody was diluted for total concentration in PBScontaining 0.02% bovine serum albumin (BSA).

Cell staining was performed using 200 μL of cell suspension comprising10⁵ cells for 20 min at room temperature. After two washing steps withPBS/0.02% BSA, the cells were resuspended in 200 μL incubated with anAPC-conjugated donkey anti-mouse IgG (H+L) secondary antibody (JacksonLaboratories, Cat. no. 715-136-150) at concentration 2 μg/mL for 20 minat room temperature. The stained cells were washed twice with PBS/0.02%BSA and kept on ice before analysis using a BD LSR Fortessa cellanalyzer (BD Biosciences).

Example 2: Mouse Monoclonal Antibodies 10C9 and 10G5 do not Cross-Reactwith Other Members of Human Tam Receptor Family

All binding experiments were performed using Biacore 3000 instrument (GEHealthcare) at 25° C. Soluble recombinant antigens corresponding to theextracellular domains of members of the human TAM receptor family, Axl(rhAxl-Fc chimera; R&D Systems, Cat. no. 154-AL), Mer (rhMer-Fc chimera;R&D Systems, Cat. no. 891-MR) and Tyro3 (rhTyro3/Dtk-Fc chimera; R&DSystems, Cat. no. 859-DK) were immobilized on the surface of CM5 sensorchip using amine coupling at the surface density of 393.0, 303.6 and364.0 resonance units (RU), respectively. The Biacore run was performedin an automatic mode using Binding to analysis wizard. Samplescontaining either MAb 10C9 or MAb 10G5 at concentration 10 μg/mL inHBS-EP buffer (GE Healthcare) were injected over the surfaces withimmobilized antigens at flow rate of 30 μL/min for 3 min (association)followed by 5 min dissociation.

The results shown in FIG. 1 demonstrate specific binding of the mousemonoclonal antibodies 10C9 and 10G5 to human Axl and no binding torecombinant human Mer and Tyro3 antigens.

Example 3: Mouse Monoclonal Antibodies 10C9 and 10G5 do not Cross-Reactwith Mouse Axl

The binding experiments were performed using Biacore 3000 instrument (GEHealthcare) at 25° C. The soluble recombinant antigens corresponding tohuman Axl (rhAxl-Fc chimera; R&D Systems, Cat. no. 154-AL), mouse Axl(rmAxl-Fc chimera; R&D Systems, R&D Systems; Cat. no. 854-AX) and humanTyro3 (rhTyro3/Dtk-Fc chimera; R&D Systems, Cat. no. 859-DK) wereimmobilized on the surface of CM5 sensor chip using amine coupling atthe surface density of 1,308.0, 2,115.9 and 1,429.0 RU, respectively.The Biacore runs were performed in an automatic mode using Bindinganalysis wizard.

The sample containing either MAb 10C9, MAb 10G5 or recombinant mouse(rm) Axl-ligand Gas6 (R&D Systems, Cat. no. 986-GS/CF) at concentration10 μg/mL in HBS-EP buffer (GE Healthcare) was injected over the surfaceswith immobilized antigens at flow rate of 30 μL/min for 3 min(association) followed by 5 min dissociation.

The results shown in FIG. 2 demonstrate specific interaction of MAbs10C9 and 10G5 with human Axl and no binding to recombinant mouse Axl andhuman Mer antigens (FIG. 2 , upper and middle panel, respectively). Incontrast, mouse Gas6, used as a control, demonstrated strong binding toboth human and mouse Axl and somewhat weaker binding to human Tyro3(FIG. 2 , lower panel).

Example 4: Mouse Monoclonal Antibodies 10C9 and 10G5 Specifically Bindto Axl Receptor from Non-Human Primates

The sequence of Axl receptor from cynomolgus monkey (Macacefascicularis; SEQ ID NO.43) was retrieved from WO2009062690A1. Based onthe sequence, recombinant extracellular domain of cyno-Axl was generatedby transient expression in CHO cells as a fusion protein with human Fc.The recombinant cyno-Axl-Fc was purified to homogeneity using ProteinA-Sepharose (GE Healthcare). The binding experiments were performedusing Biacore 3000 instrument (GE Healthcare) at 25° C. The solublerecombinant antigens corresponding to human Axl (rhAxl-Fc chimera; R&DSystems, Cat. no. 154-AL) and cyno-Axl were immobilized on the surfaceof CM5 sensor chip using amine coupling at the surface density of 775and 880 RU, respectively. The Biacore runs were performed in anautomatic mode using Binding analysis wizard.

The sample containing either MAb 10C9, MAb 10G5 or human Axl-specificMAb 5F11 (control) at concentration 10 μg/mL in HBS-EP buffer (GEHealthcare) was injected over the surfaces with immobilized antigens atflow rate of 30 μL/min for 3 min (association) followed by 5 mindissociation.

The results shown in FIG. 3 demonstrate strong and specific interactionof MAbs 10C9 and 10G5 with Axl antigens from both human and cynomolgusmonkey. In contrast, the control antibody 5F11 showed strong binding tohuman Axl and lack of cross-reactivity with Ax from cynomolgus monkey.

Example 5: Affinity Determination of Mouse Monoclonal Antibodies 10C9and 10G5

Affinity determination of anti-Axl antibodies 10C9 and 10G5 wasperformed at 25° C. by surface plasmon resonance measurements usingBiacore 3000 instrument (GE Healthcare). As a solid antigen-coatedsurface, a sensor chip CM5 with immobilized rhAxl-Fc chimera (R&DSystems, Cat. no. 154-AL) at density 190 RU was used.

For the kinetics measurements, different concentrations of anti-Axlantibodies (from 0.3 to 666.7 nM) in HBS-EP buffer (Biacore, Cat. no.BR-1001-88) were injected at flow rate of 30 μL/min with 3 min injectiontime followed by 5 min dissociation (buffer alone). After each cycle,the surface was regenerated by 30 sec injection of a regenerationsolution (10 mM HCl, 1 M NaCl) at flow rate 50 μL/min.

The mass transfer control experiments demonstrated absence ofsignificant mass transfer limitations for both used MAbs 10C9 and 10G5.An additional, linked reactions control experiment did not reveal linkedreactions for both antibodies, since the dissociation phases werepractically identical after injection for 1, 3 or 20 min of one analyteconcentration (790 nM and 160 nM for MAbs 10C9 and 10G5, respectively).

The kinetic association (on-rate, k) and dissociation (off-rate,k_(off)) rates were calculated using BIAevaluation software and 1:1Langmuir binding model. The equilibrium dissociation constant (K_(D))was calculated as the k_(off)/k_(on) ratio. The half-life (t_(1/2)) ofthe formed antibody-antigen complexes was calculated as the In2/k_(off)ratio.

As shown in FIG. 4 , the mouse MAbs 10C9 and 10G5 demonstrated highaffinities in subnanomolar range, with K_(D) values of 0.18 nM and 0.53nM, respectively.

Example 6: Mouse Monoclonal Antibodies 10C9 and 10G5 Block Binding ofGas6 to Axl

A competitive binding study was performed using Biacore 3000 instrument(GE Healthcare) and Binding Analysis wizard with several cycles of twosamples injection. As a first sample, a saturating concentration of MAb10C9 (790 nM or 120 μg/mL) or 10G5 (160 nM or 24 μg/mL) was injectedover the surface of a CM5 sensor chip coated with rhAxl-Fc (using aminecoupling) for 3 min at flow rate of 30 μL/min followed by 2.5 minstabilization (HBS-EP buffer alone) before the injection of the secondsample. The following second samples were used: recombinant human (rh)Gas6 (R&D Systems, Cat. no. 885-GS), recombinant mouse (rm) Gas6 (R&DSystems, Cat. no. 986-GS/CF) and a panel of anti-Axl antibodies, such asMAB154 (R&D Systems, Cat. no. MAB154), 10C9 and 10G5; all atconcentration 25 μg/mL. The second sample was injected for 3 min,followed by 2.5 min stabilization (buffer alone) and regeneration of thesurface by 30 sec injection of a regeneration solution (10 mM HCl, 1 MNaCl) at flow rate 50 μL/min.

The results shown in FIG. 5 demonstrated both MAbs 10C9 and 10G5 did notcompete for Axl binding with the commercial control antibody MAB154 (R&DSystems). However, the antibodies 10C9 and 10G5 blocked binding of eachother and, in addition, inhibited Axl binding by its ligand Gas6, bothof human and murine origin.

Example 7: Mouse Monoclonal Antibodies 10C9 and 10G5 Inhibit Growth ofHighly Aggressive Breast Carcinoma Cells in Tree-Dimensional (3D)Organotypic Models

A highly aggressive triple-negative human breast cancer cell lineMDA-MB-231 (ATCC® HTB-26™) was cultured according to recommendedconditions in Dulbecco's Modified Eagle's Medium/Nutrient Mixture F-12Ham medium supplemented with 10% foetal bovine serum (FBS), glutamineand penicillin and streptomycin. The cells were pre-treated insuspension for at least 1 hour at 37° C., to ensure proper binding ofantibodies on the cell surface before they were placed in extracellularmatrix. The cells cultures were observed to every day and freshtreatments were done every other day. The antibodies were used atconcentrations 50-100 μg/mL. Imaging of coverslip 3D assay (35 mm dish)was done on a NIKON light microscopy using both Phase contrast andHoffman optics. Already at day 3, difference in growth of cells treatedwith either MAb 10C9 or MAb10G5 and cells treated with a controlirrelevant IgG was observed. At day 6, it became evident that cellstreated with antibodies 10C9 or 10G5 had significantly inhibited growthand tumour mass development in the extracellular matrix, as compared tothe control-treated cells (FIG. 6 ). Nuclei staining revealed that thecells treated with MAb 10G5, despite the inhibited growth, are stillviable. Similar effect was observed for the antibody 10C9. Thisexperiment demonstrated that both anti-Axl antibodies 10C9 and 10G5 havethe potential to inhibit development of organotypic tumour masses.

Example 8: Antibodies 10C9 and 10G5 Induce Changes in Morphology of 3DTumour Colonies In Vitro

MDA-MB-231 cells were grown on extracellular matrix and allowed to formthe highly aggressive stellate shaped morphology. Stellate-shaped tumourmasses were then treated with the control IgG and antibodies 10C9 and10G5, as described in EXAMPLE 7. Both antibodies 10C9 and 10G5 causeddegradation of stellate patterns (FIG. 7 ) accompanied by cell death andDNA fragmentation. These results demonstrated that blocking Axl usingspecific monoclonal antibodies 10C9 and 10G5 has strong anti-tumoureffect in 3D models in vitro.

Example 9: Antibodies 10C9 and 10G5 Induce Axl Receptor Internalization

The expression of Axl receptor protein in MBA-MD-231 cells treated withdifferent antibodies was examined by Western blot analysis. The cellswere seeded in a 6-well plate at density of 5×10⁵ cells per well andcultured overnight before treatment initiation. The cells were treatedfor 20 hrs in the presence of isotype control (mouse IgG2b), anti-Axlantibodies (10C9, 10G5 and MAb #3) at concentrations of 100 μg/mL ormultikinase inhibitor Foretinib (targets Met, Ron, Axl, Tie-2, andVEGFR2) at a concentration of 0.5 μM followed by harvesting bycentrifugation at 1,200 rpm for 5 min and washing with sterile PBS. Thecells were collected by centrifugation and resuspended in NP40-lysisbuffer followed by 30 min incubation on ice. The cell lysates werecleared by centrifugation (12,000 rpm, 4° C., 5 min) and the proteinconcentrations were determined using BCA protein assay. The cell lysatesamples comprising 35 μg of total protein were denatured in presence ofthe reducing agent (Life Technologies) and loaded into the wells ofNuPAGE 10% Bis-Tris polyacrylamide (PAA) gel, 1.0 mm×12 well(Invitrogen). The electrophoresis was performed using Bis-Tris SDSrunning buffer under the recommended conditions (Life Technologies) andthe proteins were transfer on PVDF membrane, as described for 2 gels ina manual for XCell II Blot Module (Invitrogen) using the transfer bufferwith 20% methanol. The membrane was incubated in 10 mL of blockingbuffer, TBS/0.1% Tween20 (TBST) with 5% skimmed milk, for 1 hr at roomtemperature followed by overnight incubation in 5 mL of incubationbuffer (TBST with 3% skimmed milk) containing 1:1000 dilution ofanti-Axl MAb154 (R&D Systems) at 4° C. The membrane was washed threetimes for 5 min each with 10 mL of TBST followed by 1 hr incubation withgoat-anti mouse IgG (H+L) HRP-conjugated secondary antibody (1:2000) in5 mL of incubation buffer with gentle rolling at room temperature.Afterwards, the membrane was washed three times for 5 min in 10 mL ofTBST and twice with 10 mL of TBS buffer. The membrane was incubated with1 mL ECL substrate for 1 min at room temperature. Excess substratesolution was aspirated and the blot was visualised using a ChemiDoc™XRS+ imager (Bio Rad) and Image lab software. As loading control,detection using anti-mouse actin antibody (1:10,000; Sigma) was usedunder the same conditions.

The results shown in FIG. 8 demonstrated significant reduction of Axlprotein in cells treated with MAbs 10C9 and 10G5 compared to the cellstreated with either irrelevant IgG or MAb #3. The results indicate thatMAbs 10C9 and 10G5 induce internalization and intracellular degradationof Axl receptor.

Example 10: Antibodies 10C9 and 10G5 Block Ligand-Induced Axl DownstreamSignaling

The experiments were performed using human cervical cancer derived cellline HeLa (ATCC® CCL-2M). The cells were grown in T175 flasks to 80%confluency in MEM culture medium (Sigma) supplemented with 10% FBS,penicillin-streptomycin and L-glutamine. The cells were washed with PBSand detached by treatment with 0.25% Trypsin/EDTA (Sigma) followed bycentrifugation and resuspension in fresh medium (MEM/0.5% FBS). Thecells were seeded in Petri dishes (3×10⁶ cells per dish) in MEM mediumsupplemented with 10% FBS. After three hrs incubation at 37° C., thecells were washed with PBS and kept in starvation medium (MEM/0.5% FBS)overnight. The cells were pre-incubated with anti-Ax antibody 10C9 or10G5 at concentration 1 μg/mL for 1 hr followed by stimulation with Axlligand, recombinant mouse Gas6 (R&D Systems), at concentration 10 μg/mLfor 20 min. The cell lysates were prepared, as described in EXAMPLE 9,the Western blot analysis was performed using anti-phospho-Akt (Ser⁴⁷³)antibody (Cell Signaling) followed by goat anti-rabbit horseradishperoxidase (Jackson ImmunoResearch); the anti-phospho-Akt does notdistinguish between AKT1, AKT2, and AKT3, hence the total level of‘phospho-Akt’ is shown in the blot. Detection with anti-GAPDH antibody(Millipore) was used as loading control.

The results shown in FIG. 9 demonstrated that Axl-specific ligand Gas6induced strong Axl signalling in HeLa cells that used downstreamphosphorylation of Aid on Ser⁴⁷³ as the readout. This signalling couldsignificantly be reduced in the presence of antibody 10C9. Similarresults were obtained for the antibody 10G5.

Example 11: Sequencing of Mouse Monoclonal Antibodies 10C9 and 10G5

The hybridoma cells were propagated under standard conditions; 5×10⁶hybridoma cells were used for mRNA isolation and cDNA synthesisaccording to standard protocols. For PCR amplification of the genesencoding heavy and light chain variable regions (VH and VL,respectively), Mouse IgG Library Primer Set (Progen, Heidelberg,Germany, Cat. no. F2010) was used. For the hybridoma 10C9, PCRamplification using different primer combinations resulted in 9sequences from PCR using 5 different primer combinations for the VH geneand in 5 sequences from PCR using 2 different primer combinations forthe VL gene. The sequences of the clones VH1 (A7-1) and Vκ2 (E2-2) wereselected for further work on the basis of highest homology with thecorresponding germline sequences, as determined by nucleotide alignmentwith IMGT database.

For the hybridoma 10G5, PCR amplification using different primercombinations resulted in 12 sequences from PCR using 6 different primercombinations for the VH gene and in 5 sequences from PCR using 2different primer combinations for the VL gene. The sequences of theclones VH 1 (B6-4) and Vκ1 (F1-3) were selected for further work on thebasis of highest homology with the corresponding germline sequences, asdetermined by nucleotide alignment with IMGT database.

The deduced amino acid sequences of the VH and VL domains for antibodies10C9 and 10G5 are shown in FIG. 10 . Sequence analysis revealed thepresence of a potential N-glycosylation site in CDR1 of the heavy chain(CDR-H1; glycosylation site of ‘NFT’ is shown in bold lettering in FIG.10 )).

Also included in FIG. 10 is the sequence of a 10G5 VH variant whereinthe glutamine (Q) at position 1 of the VH domain is substituted with aglutamate (E); this variant is termed “10G5 [Q1E]”.

Example 12: Generation and Testing Chimeric Monoclonal Antibodies 10C9and 10G5

The VH and VL sequences retrieved from the murine hybridomas 10C9 and10G5 were used for generation of the synthetic genes with codonoptimization for expression in mammalian cells (GeneArt). These mouse VHand VL genes were ligated in frame with the genetic elements encodingconstant domains of the human IgG1 heavy and light (C-kappa) chains,respectively, in an expression vector suitable for antibody productionin mammalian cells. Production of the chimeric (mouse variable/humanconstant) IgG1 antibodies was achieved by transient expression inChinese Hamster Ovary (CHO) cells followed by purification using ProteinA affinity chromatography.

The purified chimeric antibodies (>95% purity) were analysed for bindingto Axl-positive breast cancer cell line MDA-MB-231 in flow cytometry.For comparison, the parental mouse MAbs 10C9 and 10G5 were used. Forflow cytometry, the adherent cells in culture were washed with PBS,detached by treatment with trypsin (0.25%) for 1 min and hitting culturedish for full detachment. Trypsin was quenched by adding into the tissueflask the complete medium followed by washing the cells with PBS. Duringthe washing steps, the cells were collected by centrifugation at 200 gfor 5 min. The antibody was diluted for total concentration in PBScontaining 0.02% bovine serum albumin (BSA). Cell staining was performedusing 200 μL of cell suspension comprising 10⁵ cells for 20 min at roomtemperature. The cell-bound antibodies were detected with APC-conjugateddonkey anti-human or anti-mouse, respectively, IgG (H+L) F(ab′)₂fragments (Jackson ImmunoResearch). After two washing steps withPBS/0.02% BSA, the cells were resuspended in 200 μL and kept on icebefore analysis on Accuri C6 flow cytometer (BD Biosciences). Theresults shown in FIG. 11 demonstrated strong binding of the chimericantibodies to the Axl-positive MDA-MB-231 cells in flow cytometry.

In addition, the Axl-binding properties of the chimeric antibodies c10C9and c10G5 were tested using Biacore 3000 instrument (GE Healthcare) anda sensor chip CM5 coated with human Axl (rhAxl-Fc chimera; R&D Systems,Cat. no. 154-AL) at the surface density of 1,308.0 RU. The Biacore runswere performed in an automatic mode using Binding analysis wizard. Thesamples comprising either the chimeric antibodies c10C9 and c10G5 ortheir murine counterparts at concentration 10 μg/mL in HBS-EP buffer (GEHealthcare) were injected over the surfaces with immobilized antigens atflow rate of 30 μL/min for 3 min (association) followed by 5 mindissociation.

The results shown in FIG. 12 demonstrate that both chimeric antibodiesc10C9 and c10G5 bind immobilized Axl with profiles very similar to thebinding profiles of the corresponding mouse antibodies derived from thehybridomas 10C9 and 10G5, respectively.

Example 13: Chimeric Antibodies 10C9 and 10G5 Bind Axl with the SameAffinities as the Parental Mouse Antibodies

Affinity determination of the chimeric anti-Axl antibodies c10C9 andc10G5 was performed at 25° C. by surface plasmon resonance measurementsusing Biacore 3000 instrument (GE Healthcare). As a solid antigen-coatedsurface, a sensor chip CM5 with immobilized rhAxl-Fc chimera (R&DSystems, Cat. no. 154-AL) at density 190 RU was used.

For the kinetics measurements, different concentrations of anti-Axlantibodies (from 0.3 to 333.3 nM) in HBS-EP buffer (Biacore, Cat. no.BR-1001-88) were injected at flow rate of 30 μL/min with 3 min injectiontime followed by 5 min dissociation (buffer alone). After each cycle,the surface was regenerated by 30 sec injection of a regenerationsolution (10 mM HCl, 1 M NaCl) at flow rate 50 μL/min.

The mass transfer control experiments demonstrated absence ofsignificant mass transfer limitations for both chimeric MAbs c10C9 andc10G5.

The kinetic association (on-rate, k_(on)) and dissociation (off-rate,k_(off)) rates were calculated using BIAevaluation software and 1:1Langmuir binding model. The equilibrium dissociation constant (K_(D))was calculated as the k_(on)/k_(off) ratio. The half-life (t_(1/2)) ofthe formed antibody-antigen complexes was calculated as the In2/k_(off)ratio.

As shown in FIG. 13 , both chimeric MAbs c10C9 and c10G5 demonstratedhigh affinities in subnanomolar range, with K_(D) values of 0.10 nM,somewhat better than the affinities of the parental murine antibodies(see EXAMPLE 5).

Example 14: Chimeric Antibody 10G5 Inhibits Tumor Growth in a MouseModel of Human Non-Small Cell Lung Cancer

To evaluate the anti-tumour activity of anti-Axl chimeric antibodies invivo, we used a mouse xenograft model of human non-small cell lungcancer (NSCLC). The human NSCLC A549 cells (ATCC #CCL-185) A549 cellswere propagated in vitro as a monolayer culture in DMEM mediumsupplemented with 10% FBS, 2 mM L-Glutamine, 100 U/ml penicillin and 100μg/ml streptomycin, 0.01M HEPES buffer, 0.45% D-(+)-glucose, 1 mM sodiumpyruvate. Nude mice were implanted subcutaneously (s.c.) into the flankwith 5×10⁶ A549 cells resuspended in serum-free medium/Matrigel (1:1).When the tumour size reached 100 mm³ (Day 0 in FIG. 14 ), the animalswere randomized and treated with either vehicle (sterile PBS) oranti-Axl chimeric antibody 10G5 at 20 mg/kg, by intraperitoneal (i.p.)injections twice weekly for 4 weeks.

As shown in FIG. 14 , the chimeric antibody 10G5 significantlyattenuated growth of A549 tumours compared with the control (P<0.01, asdetermined by two-way ANOVA); around 40% inhibition was observed afterfour weeks of treatment.

Example 15: Chimeric Antibody 10G5 Inhibits Tumor Growth in a MouseXenograft Model of Human Acute Myeloid Leukaemia

To evaluate the anti-tumour activity of anti-Axl chimeric antibodies ina model of haematological cancer, we used a mouse xenograft model ofhuman acute myeloid leukaemia (AML). The human AML Mv4-11 cells (ATCC#CRL-9591) cells were propagated in suspension in IMDM mediumsupplemented with 10% FBS, 2 mM L-Glutamine, 100 U/ml penicillin and 100μg/ml streptomycin. Nude mice were implanted s.c. into the flank with5×10⁶ Mv4-11 cells resuspended in the mixture of serum-free IMDM mediumand Matrigel (1:1). When the tumour size reached 200 mm³ (Day 0 in FIG.15 ), the animals were randomized and treated with either vehicle(sterile PBS) or anti-Axl chimeric antibody 10G5 at 30 mg/kg, by i.p.injections twice weekly for 4 weeks.

As shown in FIG. 15 , the chimeric antibody 10G5 extremely significantlyattenuated growth of Mv4-11 tumours compared with the control (P<0.0001,as determined by two-way ANOVA); around 75% inhibition was observedafter three weeks of treatment.

Example 16: Defucosylated Glycoengineered C10G5 (Glymax) Shows EnhancedAnti-Tumor Effect Compared to C10Gs in a Mouse Model of Human Non-SmallCell Lung Cancer

The naked anti-Axl antibodies can prevent tumor growth both byinhibiting the specific signaling pathway of the target receptor and/orthrough tumor cell killing via its effector functions, such asantibody-dependent cellular cytotoxicity (ADCC), complement-dependentcytotoxicity (CDC) and/or antibody-dependent cellular phagocytosis(ADCP). Antibodies lacking core fucosylation show a significantlyenhanced antibody-dependent cell-mediated cytotoxicity (ADCC) and anincreased efficacy of anti-tumor activity.

To compare the anti-tumor effects of two variants of the chimericantibody c10G5-wt and defucosylated—we used a mouse xenograft model ofhuman non-small cell lung cancer (NSCLC). The human NSCLC A549 cells(ATCC #CCL-185) A549 cells were propagated in vitro as a monolayerculture in DMEM medium supplemented with 10% FBS, 2 mM L-Glutamine, 100U/ml penicillin and 100 μg/ml streptomycin, 0.01M HEPES buffer, 0.45%D-(+)-glucose, 1 mM sodium pyruvate. SCID mice were implantedsubcutaneously (s.c.) into the flank with 5×10⁶ A549 cells resuspendedin serum-free medium/Matrigel (1:1). When the tumour size reached 130mm³ (Day 0 in FIG. 15 ), the animals were randomized and treated witheither anti-Axl c10G5 or Glymax-c10G5 at 30 mg/kg, by intraperitoneal(i.p.) injections twice weekly for 4 weeks.

As shown in FIG. 16 , the antibody Glymax-c10G5 significantly attenuatedgrowth of A549 tumours compared with the c10G5 (P<0.0001, as determinedby two-way ANOVA). The significant difference in activity of wt anddefucosylated versions of the chimeric 10G5 indicates importance ofantibody-dependent cellular cytotoxicity (ADCC) in inhibition of tumorgrowth.

Example 17: Framework Variant 1 (Fv1) Inhibits Tumor Growth in a MouseModel of Human Non-Small Cell Lung Cancer

FV1 is an antibody with the CDRs and binding specificity of 10G5, butwith multiple substitutions in the V-domain framework regions. Toevaluate the anti-tumour activity of anti-Axl FV1 in vivo, we used amouse xenograft model of human non-small cell lung cancer (NSCLC). Thehuman NSCLC A549 cells (ATCC #CCL-185) A549 cells were propagated invitro as a monolayer culture in DMEM medium supplemented with 10% FBS, 2mM L-Glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin, 0.01MHEPES buffer, 0.45% D-(+)-glucose, 1 mM sodium pyruvate. SCID mice wereimplanted subcutaneously (s.c.) into the flank with 5×10⁶ A549 cellsresuspended in serum-free medium/Matrigel (1:1). When the tumour sizereached 100 mm³ (Day 18 in FIG. 16 ), the animals were randomized andtreated with either vehicle (SYNAGIS) or anti-Axl FV1 at 30 mg/kg, byintraperitoneal (i.p.) injections twice weekly for 2 weeks.

As shown in FIG. 17 , the antibody FV1 significantly attenuated growthof A549 tumours compared with the control (P<0.051, as determined bytwo-way ANOVA); around 25% inhibition was observed after two weeks oftreatment.

Example 18: Glycoengineered Fv2 (Fv2-Glymaxx) Potentiates the Effect ofAnti-EGFR Treatment on Tumor Growth in a Mouse Model of Human Non-SmallCell Lung Cancer

FV2-GLYMAXX is a defucosylated antibody with the CDRs and bindingspecificity of 10G5, but with multiple substitutions in the V-domainframework regions. To evaluate the anti-tumour activity of FV2-GLYMAXXin vivo, we used a mouse xenograft model of human non-small cell lungcancer (NSCLC). The human NSCLC A549 cells (ATCC #CCL-185) A549 cellswere propagated in vitro as a monolayer culture in DMEM mediumsupplemented with 10% FBS, 2 mM L-Glutamine, 100 U/ml penicillin and 100μg/ml streptomycin, 0.01M HEPES buffer, 0.45% D-(+)-glucose, 1 mM sodiumpyruvate. NUDE mice were implanted subcutaneously (s.c.) into the flankwith 5×10⁶ A549 cells resuspended in serum-free medium/Matrigel (1:1).When the tumour size reached 100 mm³ (Day 0 in FIG. 18 ), the animalswere randomized and treated either with vehicle (SYNAGIS), Erbitux (20mg/kg) or FV2-GLYMAXX (15 or 30 mg/kg either alone or in combination.Antibodies were administered by intraperitoneal (i.p.) injections twiceweekly for 3 weeks.

As shown in FIG. 18 , FV2-GLYMAXX showed moderate anti-tumor activityvery similar to the anti-tumor effect of the Anti-EGFR therapeuticantibody cetuximab (Erbitux). For both antibodies used as the singleagents, the observed effect was however statistically not significantwhen compared to the mouse cohort treated with the isotype controlantibody (Synagis). Combination of both antibodies resulted insignificant tumor growth retardation (P<0.0001; as determined by two-wayANOVA) when compared to isotype control treated animals. The effect wasalso significant when compared to the groups treated with eitherFV2-GLYMAXX antibody or Erbitux alone (P<0.05; as determined by two-wayANOVA).

SEQUENCES SEQ ID NO. 1 [10C9 VH domain (nt)]CAGGTCCAACTGCAGCAGCCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGCAAGACTTCTGACTACAATTTCACACGCTACTATATACACTGGGTGAAGCAGAGGCCTGGACAGGGACTTGAGTGGATTGGATGGATTTATCCTGGAACTGGTGATTCTAAATACAATGAGAAGTTCAAGGGCAGGGCCACACTGACGGCAGACACATCCTCCAGCACTGCCTACATGCAGCTCAGCTCCCAAACATCTGAGGACTCTGCGGTCTATTTCTGTGCAAGGAATGGTAACTACTGGTACTTCGATGTCTGGGGCGCAGGGACCGCGGTCACCGTCTCCTCAGCCAAAACGACACCC SEQ ID NO. 2 [10C9 VL domain (nt)]GATATTGTGATGACGCAGGCTGCACCCTCTGGACCTGTCACTCCTGGAGAGTCAGTATCCATCTCCTGCAGGTCTAGTAAGAGTCTCCTGCATAGCAATGGCAACACTTACTTATATTGGTTCCTGCAGAGGCCAGGCCAGTCTCCTCAACTCCTGATATATCGGATGTCCAACCTTGCCTCAGGAGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGAACTGCTTTCACACTGAGAATCAGTAGAGTGGAGGCTGAGGATGTGGGTATCTATTACTGTATGCAACATCGAGAATATCCTTTCACGTTCGGAGGGGGGACCAAACTGGAAATAAAACGGGCTGATGCTGCAC CAACTGTATCCSEQ ID NO. 3 [10C9 VH domain (aa)]QVQLQQPGPELVKPGASVKISCKTSDYNFTRYYIHWVKQRPGQGLEWIGWIYPGTGDSKYNEKFKGRATLTADTSSSTAYMQLSSQTSEDSAVYFCARNGNYWYFDVWGAGTAVTVSSSEQ ID NO. 4 [10C9 VL domain (aa)]DIVMTQAAPSGPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGIYYCMQHREYPFTFGGGTKLEIKSEQ ID NO. 5 [10C9 Heavy CDR1] DYNFTRYYIH SEQ ID NO. 6 [10C9 Heavy CDR2]WIYPGTGDSKYNEKFKG SEQ ID NO. 7 [10C9 Heavy CDR3] NGNYWYFDVSEQ ID NO. 8 [10C9 Light CDR1] RSSKSLLHSNGNTYLYSEQ ID NO. 9 [10C9 Light CDR2] RMSNLAS SEQ ID NO. 10 [10C9 Light CDR3]MQHREYPFT SEQ ID NO. 11 [10C9 Heavy FR1] QVQLQQPGPELVKPGASVKISCKTSSEQ ID NO. 12 [10C9 Heavy FR2] WVKQRPGQGLEWIGSEQ ID NO. 13 [10C9 Heavy FR3] RATLTADTSSSTAYMQLSSQTSEDSAVYFCARSEQ ID NO. 14 [10C9 Heavy FR4] WGAGTAVTVSSSEQ ID NO. 15 [10C9 Light FR1] DIVMTQAAPSGPVTPGESVSISCSEQ ID NO. 16 [10C9 Light FR2] WFLQRPGQSPQLLIYSEQ ID NO. 17 [10C9 Light FR3] GVPDRFSGSGSGTAFTLRISRVEAEDVGIYYCSEQ ID NO. 18 [10C9 Light FR4] FGGGTKLEIKSEQ ID NO. 19 [10G5 VH domain (nt)]CAGGTCCAGCTGCAGCAGTCTGGGGCTGAGCTGGTGAGGCCTGGGGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACAGTTTCACTGACTTCTATATAAACTGGGTGAGGCAGAGGCCTGGACAGGGACTTGAGTGGATTGCAAGGATTTTTCCTGGAGGTGATAATACTTACTACAATGAGAAGTTCAAGGGCAAGGCCACACTGACTGCAGAAGAATCCTCCAGCACTGCCTACATACAGCTCAGCAGCCTGACATCTGAGGACTCTGCTGTCTATTTCTGTGCAAGACGGGGACTTTACTATGCTATGGACTACTGGGGTCAAGGAATCTCAGTCACCGTCTCCTCAGCCAAAACGACACCC SEQ ID NO. 20 [10G5 VL domain (nt)]GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTGCACAGTAATGGAATCCCCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAGAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACGCTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGTTCTCAAGGTACACATGTTCCTCCGACGTTCGGTGGTGGCACCAAGCTGGAAATCAAACGGGCTGATGCTGCAC CAACTGTATCCSEQ ID NO. 21 [10G5 VH domain (aa)]QVQLQQSGAELVRPGASVKLSCKASGYSFTDFYINWVRQRPGQGLEWIARIFPGGDNTYYNEKFKGKATLTAEESSSTAYIQLSSLTSEDSAVYFCARRGLYYAMDYWGQGISVTVSSSEQ ID NO. 22 [10G5 VL domain (aa)]DVLMTQTPLSLPVSLGDQASISCRSSQSLVHSNGIPYLHWYLQKPGQSPKLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQGTHVPPTFGGGTKLEIKSEQ ID NO. 23 [10G5 Heavy CDR1] GYSFTDFYINSEQ ID NO. 24 [10G5 Heavy CDR2] RIFPGGDNTYYNEKFKGSEQ ID NO. 25 [10G5 Heavy CDR3] RGLYYAMDYSEQ ID NO. 26 [10G5 Light CDR1] RSSQSLVHSNGIPYLHSEQ ID NO. 27 [10G5 Light CDR2] RVSNRFS SEQ ID NO. 28 [10G5 Light CDR3]SQGTHVPPT SEQ ID NO. 29 [10G5 Heavy FR1] QVQLQQSGAELVRPGASVKLSCKASSEQ ID NO. 30 [10G5 Heavy FR2] WVRQRPGQGLEWIASEQ ID NO. 31 [10G5 Heavy FR3] KATLTAEESSSTAYIQLSSLTSEDSAVYFCARSEQ ID NO. 32 [10G5 Heavy FR4] WGQGISVTVSSSEQ ID NO. 33 [10G5 Light FR1] DVLMTQTPLSLPVSLGDQASISCSEQ ID NO. 34 [10G5 Light FR2] WYLQKPGQSPKLLIYSEQ ID NO. 35 [10G5 Light FR3] GVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSEQ ID NO. 36 [10G5 Light FR4] FGGGTKLEIK SEQ ID NO. 37 [Human Axl]MGIQAGEPDPPEEPLTSQASVPPHQLRLGSLHPHTPYHIRVACTSSQGPSSWTHWLPVETPEGVPLGPPENISATRNGSQAFVHWQEPRAPLQGTLLGYRLAYQGQDTPEVLMDIGLRQEVTLELQGDGSVSNLTVCVAAYTAAGDGPWSLPVPLEAWRPGQAQPVHQLVKEPSTPAFSWPWWYVLLGAVVAAACVLILALFLVHRRKKETRYGEVFEPTVERGELVVRYRVRKSYSRRTTEATLNSLGISEELKEKLRDVMVDRHKVALGKTLGEGEFGAVMEGQLNQDDSILKVAVKTMKIAICTRSELEDFLSEAVCMKEFDHPNVMRLIGVCFQGSERESFPAPVVILPFMKHGDLHSFLLYSRLGDQPVYLPTQMLVKFMADIASGMEYLSTKRFIHRDLAARNCMLNENMSVCVADFGLSKKIYNGDYYRQGRIAKMPVKWIAIESLADRVYTSKSDVWSFGVTMWEIATRGQTPYPGVENSEIYDYLRQGNRLKQPADCLDGLYALMSRCWELNPQDRPSFTELREDLENTLKALPPAQEPDEILYVNMDEGGGYPEPPGAAGGADPPTQPDPKDSCSCLTAAEVHPAGRYVLCPSTTPSPAQPADRGSPAAPGQEDGA SEQ ID NO. 38 [Murine Axl]MGRVPLAWWLALCCWGCAAHKDTQTEAGSPFVGNPGNITGARGLTGTLRCELQVQGEPPEVVWLRDGQILELADNTQTQVPLGEDWQDEWKVVSQLRISALQLSDAGEYQCMVHLEGRTFVSQPGFVGLEGLPYFLEEPEDKAVPANTPFNLSCQAQGPPEPVTLLWLQDAVPLAPVTGHSSQHSLQTPGLNKTSSFSCEAHNAKGVTTSRTATITVLPQRPHHLHVVSRQPTELEVAWTPGLSGIYPLTHCNLQAVLSDDGVGIWLGKSDPPEDPLTLQVSVPPHQLRLEKLLPHTPYHIRISCSSSQGPSPWTHWLPVETTEGVPLGPPENVSAMRNGSQVLVRWQEPRVPLQGTLLGYRLAYRGQDTPEVLMDIGLTREVTLELRGDRPVANLTVSVTAYTSAGDGPWSLPVPLEPWRPVSEPPPRAFSWPWWYVLLGALVAAACVLILALFLVHRRKKETRYGEVFEPTVERGELVVRYRVRKSYSRRTTEATLNSLGISEELKEKLRDVMVDRHKVALGKTLGEGEFGAVMEGQLNQDDSILKVAVKTMKIAICTRSELEDFLSEAVCMKEFDHPNVMRLIGVCFQGSDREGFPEPVVILPFMKHGDLHSFLLYSRLGDQPVFLPTQMLVKFMADIASGMEYLSTKRFIHRDLAARNCMLNENMSVCVADFGLSKKIYNGDYYRQGRIAKMPVKWIAIESLADRVYTSKSDVWSFGVTMWEIATRGQTPYPGVENSEIYDYLRQGNRLKQPVDCLDGLYALMSRCWELNPRDRPSFAELREDLENTLKALPPAQEPDEILYVNMDEGGSHLEPRGAAGGADPPTQPDPKDSCSCLTAADVHSAGRYVLCPSTAPGPTLSADRGCPAPPGQEDGA SEQ ID NO. 39 [Human Tyro3]MALRRSMGRPGLPPLPLPPPPRLGLLLAALASLLLPESAAAGLKLMGAPVKLTVSQGQPVKLNCSVEGMEEPDIQWVKDGAVVQNLDQLYIPVSEQHWIGFLSLKSVERSDAGRYWCQVEDGGETEISQPVWLTVEGVPFFTVEPKDLAVPPNAPFQLSCEAVGPPEPVTIVWWRGTTKIGGPAPSPSVLNVTGVTQSTMFSCEAHNLKGLASSRTATVHLQALPAAPFNITVTKLSSSNASVAWMPGADGRALLQSCTVQVTQAPGGWEVLAVVVPVPPFTCLLRDLVPATNYSLRVRCANALGPSPYADWVPFQTKGLAPASAPQNLHAIRTDSGLILEWEEVIPEAPLEGPLGPYKLSWVQDNGTQDELTVEGTRANLTGWDPQKDLIVRVCVSNAVGCGPWSQPLVVSSHDRAGQQGPPHSRTSWVPVVLGVLTALVTAAALALILLRKRRKETRFGQAFDSVMARGEPAVHFRAARSFNRERPERIEATLDSLGISDELKEKLEDVLIPEQQFTLGRMLGKGEFGSVREAQLKQEDGSFVKVAVKMLKADIIASSDIEEFLREAACMKEFDHPHVAKLVGVSLRSRAKGRLPIPMVILPFMKHGDLHAFLLASRIGENPFNLPLQTLIRFMVDIACGMEYLSSRNFIHRDLAARNCMLAEDMTVCVADFGLSRKIYSGDYYRQGCASKLPVKWLALESLADNLYTVQSDVWAFGVTMWEIMTRGQTPYAGIENAEIYNYLIGGNRLKQPPECMEDVYDLMYQCWSADPKQRPSFTCLRMELENILGQLSVLSASQDPLYINIERAEEPTAGGSLELPGRDQPYSGAGDGSGMGAVGGTPSDCRYILTPGGLAEQPGQAEHQPESPLNETQRLLLLQQGLLPHSSC SEQ ID NO. 40 [Human Mer]MGPAPLPLLLGLFLPALWRRAITEAREEAKPYPLFPGPFPGSLQTDHTPLLSLPHASGYQPALMFSPTQPGRPHTGNVAIPQVTSVESKPLPPLAFKHTVGHIILSEHKGVKFNCSISVPNIYQDTTISWWKDGKELLGAHHAITQFYPDDEVTAIIASFSITSVQRSDNGSYICKMKINNEEIVSDPIYIEVQGLPHFTKQPESMNVTRNTAFNLTCQAVGPPEPVNIFWVQNSSRVNEQPEKSPSVLTVPGLTEMAVFSCEAHNDKGLTVSKGVQINIKAIPSPPTEVSIRNSTAHSILISWVPGFDGYSPFRNCSIQVKEADPLSNGSVMIFNTSALPHLYQIKQLQALANYSIGVSCMNEIGWSAVSPWILASTTEGAPSVAPLNVTVFLNESSDNVDIRWMKPPTKQQDGELVGYRISHVWQSAGISKELLEEVGQNGSRARISVQVHNATCTVRIAAVTRGGVGPFSDPVKIFIPAHGWVDYAPSSTPAPGNADPVLIIFGCFCGFILIGLILYISLAIRKRVQETKFGNAFTEEDSELVVNYIAKKSFCRRAIELTLHSLGVSEELQNKLEDVVIDRNLLILGKILGEGEFGSVMEGNLKQEDGTSLKVAVKTMKLDNSSQREIEEFLSEAACMKDFSHPNVIRLLGVCIEMSSQGIPKPMVILPFMKYGDLHTYLLYSRLETGPKHIPLQTLLKFMVDIALGMEYLSNRNFLHRDLAARNCMLRDDMTVCVADFGLSKKIYSGDYYRQGRIAKMPVKWIAIESLADRVYTSKSDVWAFGVTMWEIATRGMTPYPGVQNHEMYDYLLHGHRLKQPEDCLDELYEIMYSCWRTDPLDRPTFSVLRLQLEKLLESLPDVRNQADVIYVNTQLLESSEGLAQGSTLAPLDLNIDPDSIIASCTPRAAISVVTAEVHDSKPHEGRYILNGGSEEWEDLTSAPSAAVTAEKNSVLPGERLVRNGVSWSHSSMLPLGSSLPDELLFADDSSEGSE VLMSEQ ID NO. 41 [Human Akt3]MSDVTIVKEGWVQKRGEYIKNWRPRYFLLKTDGSFIGYKEKPQDVDLPYPLNNFSVAKCQLMKTERPKPNTFIIRCLQWTTVIERTFHVDTPEEREEWTEAIQAVADRLQRQEEERMNCSPTSQIDNIGEEEMDASTTHHKRKTMNDFDYLKLLGKGTFGKVILVREKASGKYYAMKILKKEVIIAKDEVAHTLTESRVLKNTRHPFLTSLKYSFQTKDRLCFVMEYVNGGELFFHLSRERVFSEDRTRFYGAEIVSALDYLHSGKIVYRDLKLENLMLDKDGHIKITDFGLCKEGITDAATMKTFCGTPEYLAPEVLEDNDYGRAVDWWGLGVVMYEMMCGRLPFYNQDHEKLFELILMEDIKFPRTLSSDAKSLLSGLLIKDPNKRLGGGPDDAKEIMRHSFFSGVNWQDVYDKKLVPPFKPQVTSETDTRYFDEEFTAQTITITPPEKCQQSDCGMLGNWKK SEQ ID NO. 42 [Human Gas6]MAPSLSPGPAALRRAPQLLLLLLAAECALAALLPAREATQFLRPRQRRAFQVFEEAKQGHLERECVEELCSREEAREVFENDPETDYFYPRYLDCINKYGSPYTKNSGFATCVQNLPDQCTPNPCDRKGTQACQDLMGNFFCLCKAGWGGRLCDKDVNECSQENGGCLQICHNKPGSFHCSCHSGFELSSDGRTCQDIDECADSEACGEARCKNLPGSYSCLCDEGFAYSSQEKACRDVDECLQGRCEQVCVNSPGSYTCHCDGRGGLKLSQDMDTCEDILPCVPFSVAKSVKSLYLGRMFSGTPVIRLRFKRLQPTRLVAEFDFRTFDPEGILLFAGGHQDSTWIVLALRAGRLELQLRYNGVGRVTSSGPVINHGMWQTISVEELARNLVIKVNRDAVMKIAVAGDLFQPERGLYHLNLTVGGIPFHEKDLVQPINPRLDGCMRSWNWLNGEDTTIQETVKVNTRMQCFSVTERGSFYPGSGFAFYSLDYMRTPLDVGTESTWEVEVVAHIRPAADTGVLFALWAPDLRAVPLSVALVDYHSTKKLKKQLVVLAVEHTALALMEIKVCDGQEHVVTVSLRDGEATLEVDGTRGQSEVSAAQLQERLAVLERHLRSPVLTFAGGLPDVPVTSAPVTAFYRGCMTLEVNRRLLDLDEAAYKHSDITAHSCPPVEPAAASEQ ID NO. 43 [Axl from Macaca fascicularis; also called herein″Cyno Axl″] MAWRCPRMGRVPLAWCLALCGWVCMAPRGTQAEESPFVGNPGNITGARGLTGTLRCQLQVQGEPPEVHWLRDGQILELADSTQTQVPLGEDEQDDWIVVSQLRIASLQLSDAGQYQCLVFLGHQNFVSQPGYVGLEGLPYFLEEPEDRTVAANTPFNLSCQAQGPPEPVDLLWLQDAVPLATAPGHGPQRNLHVPGLNKTSSFSCEAHNAKGVTTSRTATITVLPQQPRNLHLVSRQPTELEVAWTPGLSGIYPLTHCTLQAVLSDDGMGIQAGEPDPPEEPLTLQASVPPHQLRLGSLHPHTPYHIRVACTSSQGPSSWTHWLPVETPEGVPLGPPENISATRNGSQAFVHWQEPRAPLQGTLLGYRLAYQGQDTPEVLMDIGLRQEVTLELQGDGSVSNLTVCVAAYTAAGDGPWSLPVPLEAWRPGQAQPVHQLVKETSAPAFSWPWWYILLGAVVAAACVLILALFLVHRRKKETRYGEVFEPTVERGELVVRYRVRKSYSRRTTEATLNSLGISEELKEKLRDVMVDRHKVALGKTLGEGEFGAVMEGQLNQDDSILKVAVKTMKIAICTRSELEDFLSEAVCMKEFDHPNVMRLIGVCFQGSERESFPAPVVILPFMKHGDLHSFLLYSRLGDQPVYLPTQMLVKFMADIASGMEYLSTKRFIHRDLAARNCMLNENMSVCVADFGLSKKIYNGDYYRQGRIAKMPVKWIAIESLADRVYTSKSDVWSFGVTMWEIATRGQTPYPGVENSEIYDYLRQGNRLKQPADCLDGLYALMSRCWELNPQDRPSFTELREDLENTLKALPPAQEPDEILYVNMDEGGGYPEPPGAAGGADPPTQLDPKDSCSCLTSAEVHPAGRYVLCPSTAPSPAQPADRGSPAAPGQEDGA SEQ ID NO. 44 [Linker]GGGGS SEQ ID NO. 45 [10G5 (Q1E) VH domain (aa)]QVQLQQSGAELVRPGASVKLSCKASGYSFTDFYINWVRQRPGQGLEWIARIFPGGDNTYYNEKFKGKATLTAEESSSTAYIQLSSLTSEDSAVYFCARRGLYYAMDYWGQGISVTVSS

Biological Deposits

The present disclosure refers to two different hybridoma cell lines. Thetwo hybridoma cell lines have been deposited in accordance with the‘Budapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the Purposes of Patent Procedure’, with details asset out below.

UT-10C9-B9

-   Depositary Institution→European Collection of Cell Cultures (ECACC)    -   Public Health England    -   Porton Down    -   Salisbury    -   Wiltshire    -   SP4 OJG    -   United Kingdom-   Date of deposit→16 Dec. 2015-   Accession number→15121601-   Characteristics→Hybridoma—B-Lymphocyte; Species—M. musculus (mouse);    Morphology—lymphoblast; Immunogen—human Axl extracellular domain;    Immunocyte donor—NMRI mice; Immortal partner X63.Ag8.653; product Ig    class/sub-class-IgG2b

WR-10G5-E5

-   Depositary Institution→European Collection of Cell Cultures (ECACC)    -   Public Health England    -   Porton Down    -   Salisbury    -   Wiltshire    -   SP4 OJG    -   United Kingdom-   Date of deposit→16 Dec. 2015-   Accession number→15121602-   Characteristics→Hybridoma—B-Lymphocyte; Species—M. musculus (mouse);    Morphology—lymphoblast; Immunogen—human Axl extracellular domain;    Immunocyte donor—NMRI mice; Immortal partner X63.Ag8.653; product Ig    class/sub-class-IgG1

The invention claimed is:
 1. A method of treating a fibrotic disorder,comprising administering to a subject in need thereof an antibody thatbinds Axl, wherein the antibody comprises: A) a heavy chain variableregion (VH) that comprises a VH complementarity determining region (VHCDR) 1 with the amino acid sequence of SEQ ID NO: 5, a VH CDR2 with theamino acid sequence of SEQ ID NO: 6, and a VH CDR3 with the amino acidsequence of SEQ ID NO: 7, and a light chain variable region (VL) thatcomprises a VL complementarity determining region (VL CDR) 1 with theamino acid sequence of SEQ ID NO: 8, a VL CDR2 with the amino acidsequence of SEQ ID NO: 9, and a VL CDR3 with the amino acid sequence ofSEQ ID NO:10; or B) a VH that comprises a VH CDR 1 with the amino acidsequence of SEQ ID NO: 23, a VH CDR2 with the amino acid sequence of SEQID NO: 24, and a VH CDR3 with the amino acid sequence of SEQ ID NO: 25,and a VL that comprises a VL CDR 1 with the amino acid sequence of SEQID NO: 26, a VL CDR2 with the amino acid sequence of SEQ ID NO: 27, anda VL CDR3 with the amino acid sequence of SEQ ID NO:
 28. 2. The methodof claim 1, wherein the antibody is a humanized or a chimeric antibody.3. The method of claim 1, wherein the method comprises administering theantibody of (A), wherein the antibody of (A) comprises a VH with theamino acid sequence of SEQ ID NO: 3 and/or a VL with the amino acidsequence of SEQ ID NO:
 4. 4. The method of claim 1, wherein the methodcomprises administering the antibody of (B), wherein the antibody of (B)comprises a VH with the amino acid sequence of SEQ ID NO: 21 and/or a VLwith the amino acid sequence of SEQ ID NO:
 22. 5. The method of claim 1,wherein the method comprises administering the antibody of (A), whereinthe antibody of (A) comprises a VH with the amino acid sequence of SEQID NO: 3 and a VL with the amino acid sequence of SEQ ID NO:
 4. 6. Themethod of claim 1, wherein the method comprises administering theantibody of (B), wherein the antibody of (B) comprises a VH with theamino acid sequence of SEQ ID NO: 21 and a VL with the amino acidsequence of SEQ ID NO:
 22. 7. The method of claim 1, wherein theantibody is a whole antibody or an antigen-binding fragment.
 8. Themethod of claim 7, wherein the antibody is a monospecific, bispecific,or multispecific antigen binding fragment selected from Fv, scFv, dsFv,Fd, Fab, F(ab′)2, minibody, diabody, single-chain diabody, tandem scFv,bi-body, tri-body, or kappa(lambda)-body.
 9. The method of claim 1,wherein the antibody binds human Axl.
 10. The method of claim 1, whereinthe antibody binds Axl with a K_(D) no greater than 6×10¹⁰M.
 11. Themethod of claim 1, wherein the antibody binds Axl with a K_(D) no lowerthan 8×10⁵M⁻¹s_(−1.)
 12. The method of claim 1, wherein the antibody:(i) binds murine Axl with a K_(D) greater than 10⁻³M; (ii) binds humanMer with a K_(D) greater than 10⁻³M; and/or (iii) binds human Tyro3 witha K_(D) greater than 10⁻³M.
 13. The method of claim 1, wherein theantibody is conjugated to a detectable label, enzyme, or toxin.
 14. Themethod of claim 1, wherein the antibody is administered as animmunoconjugate comprising the antibody conjugated to a cytotoxic agent.15. The method of claim 1, wherein the antibody is administered in acomposition comprising the antibody, or an immunoconjugate thereof, anda pharmaceutically acceptable excipient.
 16. The method of claim 15,wherein the composition further comprises an immune checkpoint modulator(ICM).
 17. The method of claim 1, wherein the fibrotic disorder isstrabmisus, scleroderma, keloid, Nephrogenic systemic fibrosis,pulmonary fibrosis, idiopathic pulmonary fibrosis (IPF), cystic fibrosis(CF), systemic sclerosis, cardiac fibrosis, non-alcoholicsteatohepatitis (NASH), other types of liver fibrosis, primary biliarycirrhosis, renal fibrosis, atherosclerosis, or cancer.